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Summary Objective Early and non-invasive detection of osteoarthritis (OA) is required to enable early treatment and monitoring of interventions. Some of the earliest signs of OA are the change in proteoglycan and collagen composition. The aim of this study is to establish the relations between quantitative magnetic resonance imaging (MRI) and biochemical concentration and organization in knee articular cartilage. Methods A preregistered systematic literature review was performed using the databases PubMed and Embase. Papers were included if quantitative MRI and a biochemical assay or polarized light microscopy (PLM) was performed on knee articular cartilage, and a quantified correlation was described. The extracted correlations were pooled using a random effects model. Results 21 papers were identified. The strongest pooled correlation was found for delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) vs. proteoglycan concentration (r=0.59). T1ρ relaxation times are inversely correlated to proteoglycan concentration (r=-0.54). A weak correlation between T2 relaxation times and proteoglycans was found (r=-0.38). No correlation between T2 relaxation time and collagen concentration was found (r=-0.02). A heterogeneous set of correlations between T2 relaxation times and PLM were identified, including strong correlations to anisotropy. Conclusion DGEMRIC measures are significantly correlated to proteoglycan concentration. The needed contrast agent is however a disadvantage; the T1ρ sequence was found as a non-invasive alternative. Remarkably, no correlation was found between T2 relaxation times and collagen concentration. T2 relaxation times is related to organization, rather than concentration of collagen fibers. Prospero ID CRD42020168337.

BACKGROUND/PURPOSE Decorin is a small leucine-rich proteoglycan rich in extracellular matrix with potential antitumor activity. However, the role of decorin in hematological malignancies remains unclear, especially in the case of multiple myeloma (MM), a bone marrow (BM) stroma-dependent plasma cell neoplasm. METHODS We measured decorin levels in BM plasma samples from 270 patients with newly diagnosed MM (NDMM) using enzyme-linked immunosorbent assays. RESULTS Patients were divided into high decorin (H-DCN, > 18.99 ng/mL) and low decorin (L-DCN

Summary Objective A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) is a key enzyme in degradation of cartilage in osteoarthritis (OA). We report the pharmacological characterization of GLPG1972/S201086, a new, potent and selective small-molecule ADAMTS5 inhibitor. Methods Potency and selectivity of GLPG1972/S201086 for ADAMTS5 were determined using fluorescently-labeled peptide substrates. Inhibitory effects of GLPG1972/S201086 on interleukin-1α-stimulated glycosaminoglycan release in mouse femoral head cartilage explants, and on interleukin-1β-stimulated release of an ADAMTS5-derived aggrecan neoepitope (quantified with ELISA) in human articular cartilage explants were determined. In the destabilization of the medial meniscus (DMM) mouse and menisectomized (MNX) rat models, effects of oral GLPG1972/S201086 on relevant OA histological and histomorphometric parameters were evaluated. Results GLPG1972/S201086 inhibited human and rat ADAMTS5 (IC50 ± SD: 19 ± 2 nM and 5,000-fold selectivity over other related proteases in humans. GLPG1972/S201086 dose-dependently inhibited cytokine-stimulated aggrenolysis in mouse and human cartilage explants (100% at 20 μM and 10 μM, respectively). In DMM mice, GLPG1972/S201086 (30–120 mg/kg b.i.d) versus vehicle reduced femorotibial cartilage proteoglycan loss (23–37%), cartilage structural damage (23–39%) and subchondral bone sclerosis (21–36%). In MNX rats, GLPG1972/S201086 (10–50 mg/kg b.i.d) versus vehicle reduced cartilage damage (OARSI score reduction, 6–23%), and decreased proteoglycan loss (∼27%) and subchondral bone sclerosis (77–110%). Conclusions GLPG1972/S201086 is a potent, selective and orally available ADAMTS5 inhibitor, demonstrating significant protective efficacy on both cartilage and subchondral bone in two relevant in vivo preclinical OA models.

UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle cell attachment of C. elegans. The UNC-52 protein contains two RGD (Arg-Gly-Asp) motifs in domains III and IV, a well-characterized tripeptide known for binding to mammalian β integrin. To investigate the role of the RGD motif in UNC-52/perlecan, we created two mutations in the 2021RGD2023 motif: one mutation changed the RGD to an RGE, and the other deleted the RGD motif. The RGE2023 caused defective actin filaments and aberrant localization of PAT-3 β integrin and TLN-1/talin. Additionally, the in-frame deletion of RGD2023 resulted in a paralyzed and arrested at two-fold embryonic stages (Pat) phenotype, which is the identical phenotype of the pat-3 β integrin null allele. These results indicate that RGD2023 is a potential ligand for cell binding and is essential for development and survival. Furthermore, our analysis reveals that the RGD of an invertebrate BM molecule is a potential cell-binding motif, suggesting that the function of the RGD motif is conserved among species.

Objective The interaction between proteoglycan loss and collagen damage in articular cartilage and the effect of mechanical loading on this interaction remain unknown. The aim of this study was to answer the following questions: (1) Is proteoglycan loss dependent on the amount of collagen damage and does it depend on whether this collagen damage is superficial or internal? (2) Does repeated loading further increase the already enhanced proteoglycan loss in cartilage with collagen damage? Design Fifty-six bovine osteochondral plugs were equilibrated in phosphate-buffered saline for 24 hours, mechanically tested in compression for 8 hours, and kept in phosphate-buffered saline for another 48 hours. The mechanical tests included an overloading step to induce collagen damage, creep steps to determine tissue stiffness, and cyclic loading to induce convection. Proteoglycan release was measured before and after mechanical loading, as well as 48 hours post-loading. Collagen damage was scored histologically. Results Histology revealed different collagen damage grades after the application of mechanical overloading. After 48 hours in phosphate-buffered saline postloading, proteoglycan loss increased linearly with the amount of total collagen damage and was dependent on the presence but not the amount of internal collagen damage. In samples without collagen damage, repeated loading also resulted in increased proteoglycan loss. However, repeated loading did not further enhance the proteoglycan loss induced by damaged collagen. Conclusion Proteoglycan loss is enhanced by collagen damage and it depends on the presence of internal collagen damage. Cyclic loading stimulates proteoglycan loss in healthy cartilage but does not lead to additional loss in cartilage with damaged collagen.

OBJECTIVE To investigate the mechanism of and potential contributing factors to temporomandibular joint osteoarthritis (TMJOA) caused by oestrogen deficiency with a persistent high bite force. MATERIALS AND METHODS A TMJOA model was generated by subjecting 6-week-old female rats to ovariectomy (OVX) and feeding them a hard feed. The rats (n = 12/group) were divided into sham (control); OVX; OVX+hard feed (HF); OVX+hard feed+local-joint injection of 17β-oestradiol (an oestrogen) (E2); and OVX+hard feed+local-joint injection of rapamycin (an autophagy activator) (RAPA)groups. Condyles were stained with haematoxylin-eosin and Safranin O Fast Green. The expression of Beclin 1, LC3 and p-mTOR in condylar cartilages was analysed. RESULTS Tissue staining revealed thinner condylar cartilage, varying numbers or fewer hypertrophic chondrocytes, and lower proteoglycan content in the cartilage matrix of the OVX group. These characteristics were more pronounced in the HF group, but were significantly recovered in the E2 and RAPA groups. Immunohistochemical staining revealed significantly lower autophagic flux in OVX/HF groups and a higher one in E2/RAPA groups. CONCLUSIONS A persistent high bite force could aggravate TMJOA induced by oestrogen deficiency, and the application of oestrogen or rapamycin could delay its progression. Additionally, autophagy may play a role in the development of TMJOA.

The ECM of cartilage is composed of proteoglycans (PG) that contain glycosaminoglycan (GAG), aggrecan, hyaluronic acid (HA) and other molecular components which play an important role in regulating chondrocyte functions via cell-matrix interactions, integrin-mediated signalling etc. Implantation of chondrocytes encapsulated in scaffolds that mimic the micro-architecture of proteoglycan, is expected to enhance cartilage repair. With an aim to create a hydrogel having macromolecular structure that resembles the cartilage-specific ECM, we constructed a hierarchal structure that mimic the PG. The bottle brush structure of the aggrecan was obtained using chondroitin sulphate and carboxymethyl cellulose which served as GAG and core protein mimic respectively. A proteoglycan-like structure was obtained by cross-linking it with modified chitosan that served as a HA substitute. The physico-chemical characteristics of the above cross-linked injectable hydrogel supported long term human articular chondrocyte subsistence and excellent post-injection viability. The chondrocytes encapsulated in the PMH expressed significant levels of articular cartilage specific markers like collagen II, aggrecan, GAGs etc., indicating the ability of the hydrogel to support chondrocyte differentiation. The biocompatibility and biodegradability of the hydrogels was confirmed using suitable in vivo studies. The results revealed that the PG-mimetic hydrogel could serve as a promising scaffold for chondrocyte implantation.

OBJECTIVE This study tested the effects of small leucine-rich proteoglycan (SLRP) proteins on phosphoric acid (PA)-treated dentin bonding overtime and the role of such SLRPs in the remineralization potential of demineralized dentin collagen. METHODS Coronal dentin sections of human molars were used. SLRPs were either decorin (DCN) or biglycan (BGN) in core or proteoglycan form (with glycosaminoglycans, GAGs). Groups were: No treatment (control), DCN core, DCN + GAGs, BGN core, BGN + GAGs. Samples were etched with PA for 15 s and prior to application of Adper Single Bond Plus and composite buildup an aliquot of the specific SLRPs was applied over dentin. Twenty-four hours or 6 months after the bonding procedure, samples were tested for microtensile bond strength (MTBS). Debonded beams were analyzed by scanning electron microscopy (SEM). For remineralization studies, dentin blocks were fully demineralized, infused with the SLRPs, placed in artificial saliva for 2 weeks, and evaluated by transmission electron microscopy (TEM). RESULTS MTBS test presented a mean of 51.4 ± 9.1 MPa in control with no statistically significant difference to DCN core (47.6 ± 8.3) and BGN core (48.3 ± 6.5). The full proteoglycan groups DCN + GAGs (27.4 ± 4.5) and BGN + GAGs (36.4 ± 13.6) showed decreased MTBS compared to control (p < 0.001). At 6 months, control or core-treated samples did not have a statistically significant difference in MTBS. However, SLRPs with GAGs showed statistically significant improvement of bonding (62.5 ± 6.0 for DCN and 52.8 ± 8.1 for BGN, p < 0.001) compared to their baseline values. SEM showed that GAGs seem to favor water retention but overtime help remineralization. TEM of demineralized dentin indicated a larger collagen fibril diameter pattern of samples treated with core proteins compared to control and a smaller diameter with DCN + GAGs in water with evidence of mineralization with DCN + GAGS, BGN core and BGN + GAGs. SIGNIFICANCE In conclusion, core proteins seem not to affect dentin adhesion significantly but the presence of GAGs can be detrimental to immediate bonding. However, after ageing of samples, full proteoglycans, particularly DCN, can significantly improve bonding overtime while promoting remineralization which can prove to be clinically beneficial.

Reactive oxygen species (ROS) overproduction and oxidative stress are increasingly being implicated in the extracellular matrix (ECM) degradation associated with chronic inflammatory conditions, such as periodontal diseases. The present study investigated the effects of ROS exposure on the proteoglycans of gingival tissues, utilizing an in vitro model system comprised of supra-physiological oxidant concentrations, to ascertain whether gingival proteoglycan modification and degradation by ROS contributed to the underlying mechanisms of ECM destruction during active gingivitis. Proteoglycans were purified from ovine gingival tissues and exposed to increasing H2O2 concentrations or a hydroxyl radical (·OH) flux for 1 h or 24 h, and ROS effects on proteoglycan core proteins and sulfated glycosaminoglycan (GAG) chains were assessed. ROS were capable of degrading gingival proteoglycans, with ·OH species inducing greater degradative effects than H2O2 alone. Degradative effects were particularly manifested as amino acid modification, core protein cleavage, and GAG chain depolymerization. Proteoglycan core proteins were more susceptible to degradation than GAG chains with H2O2 alone, although core proteins and GAG chains were both extensively degraded by ·OH species. Proteoglycan exposure to ·OH species for 24 h induced significant core protein amino acid modification, with decreases in glutamate, proline, isoleucine, and leucine; and concomitant increases in serine, glycine, and alanine residues. As clinical reports have previously highlighted proteoglycan core protein degradation during chronic gingivitis, whereas their sulfated GAG chains remain relatively intact, these findings potentially provide further evidence to implicate ROS in the pathogenesis of active gingivitis, complementing the enzymic mechanisms of periodontal tissue destruction already established.

Flammulina velutipes has anti-inflammatory, immunomodulatory, antioxidant and many bioactive properties with high contents of carbohydrate, proteins and fibers. In this study, a novel proteoglycan with polysaccharide complexes and protein chain, named PGD1-1, was isolated from F. velutipes. The structural characteristics of PGD1-1 were then determined, and its anti-proliferation and pro-apoptotic activities against HepG-2 cells were demonstrated in vitro. Results proved that the average molecular weight of PGD1-1 was 32.71 kDa, and the carbohydrate and protein contents were 93.35 and 2.33%, respectively. The protein moiety was bonded to a polysaccharide chain via O-glycosidic linkage. The monosaccharides consisted of d -glucose, D-galactose and D-xylose in a molar ratio of 21.90:2.84:1.00. PGD1-1 significantly inhibited the proliferation of HepG-2 cells by affecting cell lipid peroxidation and nitric oxide production. In addition, PGD1-1 promoted the apoptosis of HepG-2 cells, especially the early apoptosis. These findings proved that PGD1-1 was a novel potent ingredient against the proliferation of HepG-2, which will provide a theoretical basis for the development and utilization of the functional ingredients of the F. velutipes.

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and an effective target for the treatment of type 2 diabetes (T2D). A natural hyperbranched proteoglycan extracted from Ganoderma lucidum, namely, Fudan-Yueyang G. Lucidum (FYGL), was demonstrated capable of inhibiting the activity of PTP1B. Here, to identify the effective active components of FYGL, three different components, the polysaccharide FYGL-1, proteoglycans FYGL-2, and FYGL-3, were isolated from FYGL, and then, the protein moiety of FYGL-3 was further separated, namely, FYGL-3-P. Their abilities to enhance the glucose uptake in cells and inhibit the activity of PTP1B were compared. The inhibitory mechanisms were systematically explored by spectroscopic methods and MD simulations. The results showed that FYGL-3 and FYGL-3-P significantly enhanced the insulin-provoked glucose uptake in insulin-resistant HepG2 cells, detected by the glucose oxidase method. Also, the FYGL-3-P protein moiety in FYGL played an essential role in inhibiting the activity of PTP1B. A strong, enthalpy-driven, and multitargeted interaction by electrostatic forces between PTP1B and FYGL-3-P dramatically inhibited the catalytic activity of PTP1B. These results provided deep insights into the molecular mechanisms of FYGL inhibiting the activity of PTP1B and structurally helped researchers seek natural PTP1B inhibitors.

Recombinant human BMP-4 growth factor (GF) has significant commercial potential as therapeutic for regenerating bone and as cell culture supplement. However, its commercial utility has been limited as large-scale attempts to express and purify human BMP-4 GF have proved challenging. We have established a novel approach to obtain significant quantities of pure and bioactive BMP-4 GF from Chinese hamster ovary cell cultures by extracting the GF moiety from the extracellular matrix or cell pellet fraction. This approach increased yields approximately one 100-fold over BMP-4 GF purified from CM. The molecular activities of the two fractions are indistinguishable. We further analyzed binding of BMP-4 GF to the proteoglycan Heparin and showed that an N-terminal basic sequence is essential for this interaction. Taken together, these results provide novel insights into the purification, localization, and Heparin binding of human BMP-4 that have implications for its bioprocessing and biological function.

The organelle of eukaryotes is a finely regulated system. Once disturbed, it activates the specific autoregulatory systems, namely, organelle autoregulation. Among which, the Golgi stress response accounts for one. When the abundance and capacity of the Golgi apparatus are insufficient compared with cellular demand, the Golgi stress response is activated to enhance the function of the Golgi apparatus. Although the molecular mechanism of the Golgi stress response has not been well characterized yet, it seems to be an important part of the mammalian stress response. In this review, we discuss the current status of research on the six pathways of the mammalian Golgi stress response (the TFE3, heat shock protein 47, CREB3, E26 transformation specific, proteoglycan, and mucin pathways), which regulate the general function of the Golgi apparatus, anti-apoptosis, pro-apoptosis, proteoglycan glycosylation, and mucin glycosylation, respectively.

Injured tendons do not regain their native structure except at fetal or very young ages. Healing tendons often show mucoid degeneration involving accumulation of sulfated glycosaminoglycans (GAGs), but its etiology and molecular base have not been studied substantially. We hypothesized that quality and quantity of gene expression involving synthesis of proteoglycans having sulfated GAGs are altered in injured tendons and that a reduction in synthesis of sulfated GAGs improves structural and functional recovery of injured tendons. C57BL6/j mice were subjected to the Achilles tendon tenotomy surgery. The injured tendons accumulated sulfate proteoglycans as early as 1-week postsurgery and continued so by 4-week postsurgery. Transcriptome analysis revealed upregulation of a wide range of proteoglycan genes that have sulfated GAGs in the injured tendons 1 and 3 weeks postsurgery. Genes critical for enzymatic reaction of initiation and elongation of chondroitin sulfate GAG chains were also upregulated. After the surgery, mice were treated with the 2-deoxy-D-glucose (2DG) that inhibits conversion of glucose to glucose-6-phosphate, an initial step of glucose metabolism as an energy source and precursors of monosaccharides of GAGs. The 2DG treatment reduced accumulation of sulfated proteoglycans, improved collagen fiber alignment and reduced the cross-sectional area of the injured tendons. The modulus of the 2DG-treated groups were higher than that in the vehicle group, but not of statistical significance. Our findings suggest that mucoid degeneration in injured tendons may result from upregulated expression of genes involved synthesis of sulfate proteoglycans and can be inhibited by reduction of glucose utilization. This article is protected by copyright. All rights reserved.

Blood compatible materials are required for the development of therapeutic and diagnostic blood contacting devices as blood-material interactions are a key factor dictating device functionality. In this work, we explored biofunctionalization of silk biomaterials with a recombinantly expressed domain V of the human basement membrane proteoglycan perlecan (rDV) towards the development of blood compatible surfaces. Perlecan and rDV are of interest in vascular device development as they uniquely support endothelial cell, while inhibiting smooth muscle cell and platelet interactions. rDV was covalently immobilized on silk biomaterials using plasma immersion ion implantation (PIII), a new method of immobilizing proteins on silk biomaterials that does not rely on modification of specific amino acids in the silk protein chain, and compared to physisorbed and carbodiimide immobilized rDV. Untreated and treated silk biomaterials were examined for interactions with blood components with varying degrees of complexity, including isolated platelets, platelet rich plasma, blood plasma, and whole blood, both under agitated and flow conditions. rDV-biofunctionalized silk biomaterials were shown to be blood compatible in terms of platelet and whole blood interactions and the PIII treatment was shown to be an effective and efficient means of covalently immobilizing rDV in its bioactive form. These biomimetic silk biomaterials are a promising platform toward development of silk-based blood-contacting devices for therapeutic, diagnostic, and research applications. STATEMENT OF SIGNIFICANCE: Blood compatible materials are required for the development of therapeutic and diagnostic blood contacting devices as blood-material interactions are a key factor dictating device functionality. In this work, we explored biofunctionalization of silk biomaterials with a recombinantly expressed domain V (rDV) of the human basement membrane proteoglycan perlecan towards the development of blood compatible surfaces. Perlecan and rDV are of interest in vascular device development as they uniquely support endothelial cell, while inhibiting smooth muscle cell and platelet interactions. rDV was covalently immobilized on silk biomaterials using plasma immersion ion implantation (PIII), a new method of immobilizing proteins on silk biomaterials that does not rely on modification of specific amino acids in the silk protein chain. These biomimetic silk biomaterials are a promising platform toward development of silk-based blood-contacting devices for therapeutic, diagnostic, and research applications.

Glycosaminoglycans (GAGs) are long, linear polysaccharides comprised of repeating disaccharide units with pleiotropic biological functions, with the non-sulfated GAG hyaluronic acid (HA), and sulfated GAGs dermatan sulfate, chondroitin sulfate, heparan sulfate, keratan sulfate, and to a lesser extent heparin all being expressed in skin. Their ability to regulate keratinocyte proliferation and differentiation, inflammatory processes and extracellular matrix composition and quality demonstrates their critical role in regulating skin physiology. Similarly, the water-binding properties of GAGs and structural qualities, particularly for HA, are crucial for maintaining proper skin form and hydration. The biological importance of GAGs, as well as extensive evidence that their properties and functions are altered in both chronological and extrinsic skin aging, makes them highly promising targets to improve cosmetic skin quality. Within the present review, we examine the cutaneous biological activity of GAGs alongside the protein complexes they form called proteoglycans and summarize the age-related changes of these molecules in skin. We also examine current topical interventional approaches to modulate GAGs for improved skin quality such as direct exogenous administration of GAGs, with a particular interest in strategies targeted at potentiating GAG levels in skin through either attenuating GAG degradation or increasing GAG production.

Regions of high mammographic density (MD) in the breast are characterised by a proteoglycan (PG)-rich fibrous stroma, where PGs mediate aligned collagen fibrils to control tissue stiffness and hence the response to mechanical forces. Literature is accumulating to support the notion that mechanical stiffness may drive PG synthesis in the breast contributing to MD. We review emerging patterns in MD and other biological settings, of a positive feedback cycle of force promoting PG synthesis, such as in articular cartilage, due to increased pressure on weight bearing joints. Furthermore, we present evidence to suggest a pro-tumorigenic effect of increased mechanical force on epithelial cells in contexts where PG-mediated, aligned collagen fibrous tissue abounds, with implications for breast cancer development attributable to high MD. Finally, we summarise means through which this positive feedback mechanism of PG synthesis may be intercepted to reduce mechanical force within tissues and thus reduce disease burden.

Highlights • Fast Field-Cycling can assess proteoglycan content in vitro. • The T1 vs field strength profile of cartilage varies in osteoarthritis. • These biomarkers can potentially be accessed in vivo using Field-Cycling Imaging. • Potential application in clinical trials of new treatments for early osteoarthritis., Osteoarthritis in synovial joints remains a major cause of long-term disability worldwide, with symptoms produced by the progressive deterioration of the articular cartilage. The earliest cartilage changes are thought to be alteration in its main protein components, namely proteoglycan and collagen. Loss of proteoglycans bound in the collagen matrix which maintain hydration and stiffness of the structure is followed by collagen degradation and loss. The development of new treatments for early osteoarthritis is limited by the lack of accurate biomarkers to assess the loss of proteoglycan. One potential biomarker is magnetic resonance imaging (MRI). We present the results of a novel MRI methodology, Fast Field-Cycling (FFC), to assess changes in critical proteins by demonstrating clear quantifiable differences in signal from normal and osteoarthritic human cartilage for in vitro measurements. We further tested proteoglycan extracted cartilage and the key components individually. Three clear signals were identified, two of which are related predominantly to the collagen component of cartilage and the third, a unique very short-lived signal, is directly related to proteoglycan content; we have not seen this in any other tissue type. In addition, we present the first volunteer human scan from our whole-body FFC scanner where articular cartilage measurements are in keeping with those we have shown in tissue samples. This new clinical imaging modality offers the prospect of non-invasive monitoring of human cartilage in vivo and hence the assessment of potential treatments for osteoarthritis. Keywords: Fast Field-Cycling NMR; human hyaline cartilage; Osteoarthritis; T1 dispersion; quadrupolar peaks; protein interactions

Zika virus (ZIKV) infection has been associated with fetal abnormalities by compromising placental integrity, but the mechanisms by which this occurs are unknown. Flavivirus can deregulate the host proteome, especially extracellular matrix (ECM) proteins. We hypothesize that a deregulation of specific ECM proteins by ZIKV, affects placental integrity. Using twelve different placental samples collected during the 2016 ZIKV Puerto Rico epidemic, we compared the proteome of five ZIKV infected samples with four uninfected controls followed by validation of most significant proteins by immunohistochemistry. Quantitative proteomics was performed using tandem mass tag TMT10plex™ Isobaric Label Reagent Set followed by Q Exactive™ Hybrid Quadrupole Orbitrap Mass Spectrometry. Identification of proteins was performed using Proteome Discoverer 2.1. Proteins were compared based on the fold change and p value using Limma software. Significant proteins pathways were analyzed using Ingenuity Pathway (IPA). TMT analysis showed that ZIKV infected placentas had 94 reviewed differentially abundant proteins, 32 more abundant, and 62 less abundant. IPA analysis results indicate that 45 of the deregulated proteins are cellular components of the ECM and 16 play a role in its structure and organization. Among the most significant proteins in ZIKV positive placenta were fibronectin, bone marrow proteoglycan, and fibrinogen. Of these, fibrinogen was further validated by immunohistochemistry in 12 additional placenta samples and found significantly increased in ZIKV infected placentas. The upregulation of this protein in the placental tissue suggests that ZIKV infection is promoting the coagulation of placental tissue and restructuration of ECM potentially affecting the integrity of the tissue and facilitating dissemination of the virus from mother to the fetus.

Objectives The function of bone is to protect the vital organs of the body. Mechanical strength, especially compressive strength, plays an important role in fulfilling its function. Fracture healing depends on several substances, such as collagen, glucosaminoglycane and proteoglycan. Chondroitin sulfate as part of proteoglycane is an important component in the formation of callus in fracture healing. The aim of this study is to prove chondroitin sulfate role in supporting fracture healing. Methods The in vivo experiment has been performed to Rattus novergicus which met the inclusion criteria (age 3 months, 200–300 g weight), 18 males of R. norvegicus, Wistar strain, were divided into three equal groups of six rats each. After being anesthetized, fracturation was performed in a sterile manner to get simple fracture. The area of dissection is in half length of tibial bone and the fracture incision is about 1 cm. Then it followed by immobilization of the lower leg bone on one side with a cast. The first group was given chondroitin sulfate 7 mg in 2 mL distilled water/200 g weight for 2 weeks. The second group was given chondroitin sulfate 7 mg in 2 mL distilled water/200 g weight for 4 weeks. The third group was given distilled water. This research was focused on treatment of cartilage. The callus position is in half length of tibial bone. Results There were significant differences in the increase of TGF-β, the number of osteoblasts and callus compressive strength in the groups with chondroitin sulfate treatment for 2 and 4 weeks, compared to the control group (p Conclusions Administering chondroitin sulfate in a dose of 7 mg in 2 mL distilled water for 2 and 4 weeks may increase production of TGF-β, the osteoblast numbers and the callus compressive strength in fracture healing.

Objective: Human endothelial cells produce 2 alternatively spliced TFPI (tissue factor pathway inhibitor) isoforms that maintain anticoagulant properties of the vasculature. TFPIβ is glycosylphosphatidylinositol anchored on the cell surface. TFPIα has a basic C terminus sharing homology with VEGF (vascular endothelial growth factor) and is a heparin-releasable protein, suggesting it binds glycosaminoglycans on the endothelium surface. However, this is unclear because TFPIα is not on the surface of cultured endothelial cells. This study identifies the source of heparin-releasable TFPIα. Approach and Results: ELISA assays localized heparin-releasable TFPIα to the extracellular matrix (ECM) of Ea.hy926 cells and human umbilical vein endothelial cells. Immunofluorescence microscopy for TFPIα showed punctate intracytoplasmic staining and ECM staining beneath individual cells. Flow cytometry identified TFPIβ but not TFPIα on the cell surface. TFPIα localization to ECM was confirmed with ELISA and immunohistochemistry studies of umbilical cord veins. The TFPIα C terminus interacted with Ea.hy926 ECM glycosaminoglycans, and a homologous VEGF peptide competed for this binding, suggesting these interactions modulate VEGF responses. Immobilized TFPIα C-terminal peptide bound to several ECM proteoglycans in Ea.hy926 conditioned media. Immunofluorescence studies of human kidney colocalized TFPIα with 4 of these proteoglycans surrounding the microvasculature: glypican-1, syndecan-4, thrombospondin, and laminin-5. The absence of TFPIα on the surface of endothelial cells and its co-localization with specific ECM proteins suggests TFPIα binds to unique proteoglycan structures. Conclusions: ECM contained the primary vascular pool of heparin-releasable TFPIα. By localizing to ECM, TFPIα is positioned to inhibit the procoagulant activity of tissue factor surrounding the vasculature.

Desmoplasia in the extracellular matrix (ECM) is one of the hallmarks of pancreatic cancer (PC), a virtually incurable disease. Decorin, a classical small leucine-rich proteoglycan found in the ECM, was upregulated in PC tissue samples according to the data of TCGA. However, decorin plays a protective role in the ECM. So it is necessary to study the roles of decorin in the progression of PC. A significantly upregulated expression of decorin was observed in the PC tissue samples compared with the normal tissues. However, there was no considerable difference in the level of expression of decorin during different pathological stages, which was supported by the immunoblot analysis. Western blot showed a higher expression of decorin A in the para-carcinoma tissue than in the cancerous tissue but the expression of decorin B, C, and D was elevated in the cancerous tissue. The results of the MTT and scratch wound healing assays revealed an elevated proliferation ability and migration rate in decorin B-overexpressing cells but were inhibited in the decorin A-overexpressing cells. Overexpression of decorin A significantly elevated the expression of the apoptosis-related genes and Decorin B-overexpression elevated proliferation-related genes. All the results showed that decorin B played important roles in the promoting of PC.

Acmella oleracea contains spilanthol as the main active compound, which possesses analgesic and anti-inflammatory effects that can favor tendon reorganization. To analyze the effect of A. oleracea on the content and organization of collagen in injured tendons, the calcaneal tendon of male Lewis rats was partially transected and treated at the site of injury with a topical application of 20% A. oleracea ointment (AO group) or with the ointment base without the plant extract (B group). The animals were euthanized 21 days after partial transection. Higher collagen concentration was observed in the AO group than in the B group, and morphological analysis using polarization microscopy showed higher birefringence in the AO group than in the B group, indicating higher collagen organization. No difference was observed in the number of fibroblasts, blood vessels, proteoglycan distribution, and maximum load between the B and AO groups. In conclusion, topical application of 20% A. oleracea ointment increased the molecular organization and content of collagen, thus indicating a potential application in tendon repair. Studies on the later phases of the tendon healing process are necessary to demonstrate the possible biomechanical changes after the application of A. oleracea ointment.

Injectable therapies for intervertebral disc (IVD) repair are gaining much interest. Recently, a chitosan (CH)-based injectable scaffold has been developed that has similar mechanical properties to human nucleus pulposus (NP) and provides a suitable environment for encapsulated NP cell survival and proteoglycan production. The hypothesis of the study was that the biological response of the encapsulated cells can be further increased by adding gelatine and Link N (LN, a naturally occurring peptide present in cartilage and IVD extracellular matrix), known to increase cell adhesion and proteoglycan production, respectively. The effect of gelatine on the mechanical properties of a CH hydrogel was evaluated through rheological and compressive mechanical tests. Production of proteoglycan [assessed as glycosaminoglycan (GAG)] by encapsulated NP cells was determined in the presence or absence of gelatine in normal or degenerative medium supplemented with LN. Normal and degenerative media replicate the healthy and degenerative disc environment, respectively. Gelatine slightly reduced the gelation rate of CH hydrogel but improved its final mechanical properties in compression. LN had a minimal effect in normal medium but induced significantly more GAG production in degenerative medium (p < 0.001, 4.7-fold superior to the control), reaching similar results to transforming growth factor (TGF)-β (used as a positive control). GAG production was further increased in CH-gelatine hydrogels, confirming an additive effect of LN and gelatine in a degenerative environment. The results supported the concept that CH-gelatine hydrogels supplemented with LN can help restore the function of the NP during the early stages of IVD degeneration.

The aim of this study was to evaluate the intensity of decorin in normal and the injured equine cadaver limbs. The study was carried out using superficial digital flexor tendon (SDFT) samples harvested from equine cadaver limbs. Randomly assigned left or right equine distal forelimbs were collected in normal (7 - 14 years old ± 2.86 SD) and injured (6 - 16 years old ± 3.64 SD) SDFT. Small pieces of approximately 5 x 5 x 3 mm were collected from normal and the injured areas at the mid-most metacarpal regions and fixed in 4% paraformaldehyde at room temperature. They were routinely prepared for H&E staining in order to confirm whether they were normal or injured. Then they were prepared for immunological assay, the primary and the secondary antibodies were prepared in a standard ratio. The results showed that the distribution of decorin was varied between fascicles and the interfascicular matrix (IFM), and the latter showed greater intensity than the fascicles. The injured tendons showed up-regulation of decorin, it was intensively expressed in all the cases of injured tendon, particularly fascicles showed significant up-regulation when compared to the normal tendon. It was concluded that decorin was varied regionally, and the injured tendons presented a significant up-regulation.

As letters form the vocabulary of a language, biochemical 'symbols' (the building blocks of oligo- and polymers) make writing molecular messages possible. Compared to nucleotides and amino acids, sugars have chemical properties that facilitate to reach an unsurpassed level of oligomer diversity. These glycans are a part of the ubiquitous cellular glycoconjugates. Cyto- and histochemically, the glycans' structural complexity is mapped by glycophenotyping of cells and tissues using receptors ('readers', thus called lectins), hereby revealing its dynamic spatiotemporal regulation: these data support the concept of a sugar code. When proceeding from work with plant (haem)agglutinins as such tools to the discovery of endogenous (tissue) lectins, it became clear that a broad panel of biological meanings can indeed be derived from the sugar-based vocabulary (the natural glycome incl. post-synthetic modifications) by glycan-lectin recognition in situ. As consequence, the immunocyto- and histochemical analysis of lectin expression is building a solid basis for the steps toward tracking down functional correlations, for example in processes leading to cell adhesion, apoptosis, autophagy or growth regulation as well as targeted delivery of glycoproteins. Introduction of labeled tissue lectins to glycan profiling assists this endeavor by detecting counterreceptor(s) in situ. Combining these tools and their applications strategically will help to take the trip toward the following long-range aim: to compile a dictionary for the glycan vocabulary that translates each message (oligosaccharide) into its bioresponse(s), that is to crack the sugar code.

Background Serglycin (SRGN) is an intragranular, sulfated proteoglycan in hematopoietic cells that affects granule composition and function. Objective To understand how SRGN affects platelet granule packaging, cargo release, and extra-platelet microenvironments. Methods Platelets and megakaryocytes from SRGN-/- mice were assayed for secretion kinetics, cargo levels, granule morphology upon activation, and receptor shedding. Results Metabolic, 35 SO4 labeling identified SRGN as a major sulfated macromolecule in megakaryocytes. SRGN colocalized with α-granule markers (platelet factor 4 [PF4], von Willebrand factor [VWF], and P-selectin), but its deletion did not affect α-granule morphology or number. Platelet α-granule composition was altered, with a reduction in basic proteins (pI ≥8; e.g., PF4, SDF-1, angiogenin) and constitutive release of PF4 from SRGN-/- megakaryocytes. P-Selectin, VWF, and fibrinogen were unaffected. Serotonin (5-HT) uptake and β-hexosaminidase (HEXB) were slightly elevated. Thrombin-induced exocytosis of PF4 from platelets was defective; however, release of RANTES/CCL5 was normal and osteopontin secretion was more rapid. Release of 5-HT and HEXB (from dense granules and lysosomes, respectively) were unaffected. Ultrastructural studies showed distinct morphologies in activated platelets. The α-granule lumen of SRGN-/- platelet had a grainy staining pattern, whereas that of wild-type granules had only fibrous material remaining. α-Granule swelling and decondensation were reduced in SRGN-/- platelets. Upon stimulation of platelets, a SRGN/PF4 complex was released in a time- and agonist-dependent manner. Shedding of GPVI from SRGN-/- platelets was modestly enhanced. Shedding of GP1b was unaffected. Conclusion The polyanionic proteoglycan SRGN influences α-granule packaging, cargo release, and shedding of platelet membrane proteins.

In order to make clear the protein compositions of Sipunculus nudus and investigate its immune-related proteins, proteomic analysis was performed on body wall and coelomic fluid of Sipunculus nudus. A total of 1659 proteins were identified, and 539 proteins were differentially expressed in the coelomic fluid compared to those in the body wall, of which 415 proteins were up-regulated while 124 proteins were down-regulated. Gene Ontology (GO) analysis showed that the GO terms involved in the two parts of Sipunculus nudus were similar, with metabolic processes, catalytic activity and cell occupying the top categories of biological process, molecular function and cellular component, respectively. KEGG pathway analysis showed that 49 pathways in body wall and 48 in coelomic fluid were mapped respectively, and these pathways were mainly related to cellular processes, environmental information processing, genetic information processing and metabolism. The COG analysis showed that 757 proteins from body wall and 889 from coelomic fluid were classified into 26 COG categories, respectively. Pfam annotation revealed the mainly immune-related proteins contained in Sipunculus nudus, such as insulin-like growth factor binding protein, catalase, basement membrane proteoglycan, titin. Our research provides the first proteomic information of Sipunculus nudus, which contributes to the study of functional proteins in Sipunculus nudus and is of great significance for the application of Sipunculus nudus in functional foods and medicines.

SLC37A4-CDG is an emerging congenital disorder of glycosylation which is characterized by a dominant inheritance and a major coagulopathy originating from the liver. Recent studies took interest in the biochemical alterations found in this CDG and showed that they consisted of multiple glycosylation abnormalities, which result from mislocalization of the endoplasmic reticulum glucose-6-phosphate transporter and associated Golgi homeostasis defects. In this work, we highlight in six affected individuals abnormal patterns for various serum N-glycoproteins and bikunin proteoglycan isoforms, together with specific alterations of the mass spectra of endoglycosidase H-released serum N-glycans. Collectively, these data complement previous findings, help to better delineate SLC37A4-CDG and could present interest in diagnosing this disease.

Little is known about extracellular matrix (ECM) contributions to formation of the earliest cell lineages in the embryo. Here, we show that the proteoglycan versican and glycosaminoglycan hyaluronan are associated with emerging Flk1+hematoendothelial progenitors at gastrulation. The mouse versican mutantVcanhdflacks yolk sac vasculature, with attenuated yolk sac hematopoiesis. CRISPR/Cas9-mediatedVcaninactivation in mouse embryonic stem cells reduced vascular endothelial and hematopoietic differentiation in embryoid bodies, which generated fewer blood colonies, and had an impaired angiogenic response to VEGF165. HA was severely depleted inVcanhdfembryos, with corresponding increase in the HA-depolymerase TMEM2. Conversely, HA-deficient mouse embryos also had vasculogenic suppression but with increased versican proteolysis. VEGF165and Indian hedgehog, crucial vasculogenic factors, utilized the versican-HA matrix, specifically versican chondroitin sulfate chains, for binding. Versican-HA ECM is an obligate requirement for vasculogenesis and primitive hematopoiesis, acts as an vasculogenic factor-enriching microniche for Flk1+progenitors from their origin at gastrulation.

Though numerous proteases have been isolated and screened for the dehairing purpose, their use in the leather industry is limited mainly due to high cost, the need for expertise, and control during unit operation and alterations in the quality of leather due to lack of the right kind of substrate specificity of the enzymes used. This paper deals with the comparative specificity and dehairing efficiency of proteases isolated from Bacillus cereus VITSP01 (PE2) and Brevibacterium luteolum VITSP02 (PE). PE2 and PE were found to be trypsin-like and elastase-like serine proteases respectively. The protease of VITSP02 degraded the proteoglycans efficiently in comparison to that of VITSP01. The results suggest that the possible targets of the studied proteases might be skin proteoglycans, including those cementing the hair root bulb. Hence, an in-depth study on the substrate specificity of the dehairing proteases would help in designing an improved screening method for isolating potent dehairing enzymes.

Pleiotrophin (PTN) is a potent mitogenic cytokine whose activities are controlled by its interactions with glycosaminoglycan (GAG). We examined the specificity of PTN for several types of GAG oligosaccharides. Our data indicate that the interaction of PTN with GAGs is dependent on the sulfation density of GAGs. Surprisingly, an acidic peptide also had similar interactions with PTN as GAGs. This shows that the interaction of PTN with anionic polymers is flexible and adaptable and that the charge density is the main determinant of the interaction. In addition, we show that PTN can compensate for the loss of its termini in interactions with heparin oligosaccharides, allowing it to maintain its affinity for GAGs in the absence of the termini. Taken together, these data provide valuable insight into the interactions of PTN with its proteoglycan receptors.

Nanoparticles can be prepared by several sophisticated processes but until now, it cannot be prepared by simple home cooking. Here, we report that two incidental food nanoparticles (iFNPs) consisting of proteoglycans and phytosterols were isolated from soup made from freshwater clam (Corbicula fluminea Muller), a renowned folk remedy for liver problems in China and other parts of East Asia. These two bioactive iFNPs were obtained and characterized by anionic exchange chromatography coupled with multi-angle laser light scattering measurement. Their hydrodynamic diameters and ζ-potentials were 50 ± 0.2 nm and -28.0 mV and 67 ± 0.4 nm and -9.96 mV, respectively. FT-IR revealed that the proteoglycans in the particles contained α-type heteropolysaccharides. Both iFNPs were resistant to pH changes and separation by mechanical force but responsive to temperature changes. They effectively inhibited cholesterol uptake in vitro, which resonates with the traditional belief that freshwater clam soup provides hepatoprotective benefits. This study suggests that these two proteoglycan-lipid iFNPs are the active moieties and offers a supramolecular structure-based approach to study the function of such complex matrices derived from food.

Osteoarthritis (OA) is the most common chronic and degenerative joint disease. Although traditional OA medications can partially relieve pain, these medications cannot completely cure OA. Therefore, it is particularly important to find an effective treatment for OA. This study explored the function of long non-coding RNA (lncRNA)-colorectal neoplasia differentially expressed gene (CRNDE) in the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the underlying molecular mechanism, aiming to develop a new treatment method for osteoarthritis. BMSCs were isolated from rat bone marrow using the gradient centrifugation method. And BMSC chondrogenic differentiation was induced with chondrogenic medium. The expression of lncRNA-CRNDE was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Silent information regulator factor 2-related enzyme 1 (SIRT1) and cartilage marker genes Aggrecan and collagen 2 (α1) protein expression were researched using western blot. Alcian blue staining was employed to examine the content of cartilage matrix proteoglycan glycosaminoglycan (GAG). The interaction between lncRNA-CRNDE and SIRT1 was detected by RNA pull-down and RNA immunoprecipitation (RIP) assay. Ubiquitination experiments were performed to measure the ubiquitination level of SIRT1. The combination between SMAD ubiquitination regulatory factor 2 (SMURF2) and SIRT1, as well as SRY-related high-mobility-group box 9 (SOX9) and collagen 2 (α1) promoter, was detected by Co-immunoprecipitation or ChIP. With the prolongation of induction time, the expression of lncRNA-CRNDE, SIRT1, cartilage marker genes Aggrecan and collagen 2 (α1) in BMSC osteogenic differentiation was gradually increased. Also, the content of cartilage matrix proteoglycan GAG was gradually elevated with the extension of the induction time. Further increase in the expression of SIRT1, cartilage marker genes Aggrecan and collagen 2 (α1) by overexpression of lncRNA-CRNDE also indicated elevated GAG content. RNA pull-down and RIP assay confirmed the binding between lncRNA-CRNDE and SIRT1. qRT-PCR and western blot showed that interference with lncRNA-CRNDE significantly inhibited the protein expression of SIRT1. BMSCs transfected with si-CRNDE increased ubiquitination levels of SIRT1 mediated by the E3 ligase SMURF2, leading to the reduced protein stability of SIRT1. However, overexpression of lncRNA-CRNDE increased the binding ability of SOX9 and collagen 2 (α1) promoter, which was reversed by the simultaneous transfection of CRNDE overexpression (pcDNA-CRNDE) and SIRT1 small interfering RNA (si-SIRT1). lncRNA-CRNDE regulates BMSC chondrogenic differentiation to promote cartilage repair in osteoarthritis through SIRT1/SOX9.

Lumican (LUM), a small leucine-rich proteoglycan, is a component of the extracellular matrix. Abnormal LUM expression is potentially associated with cancer progression. In the present study, we confirmed high LUM mRNA expression in colorectal adenocarcinoma (COAD) through the UALCAN database. The Kaplan-Meier method, univariate, and multivariate COX analysis showed that high LUM expression is an independent determinant of poor prognosis in COAD. A COX regression model was constructed based on clinical information and LUM expression. The receiver operating characteristic (ROC) curve indicated that this model was highly accurate in monitoring COAD prognosis. The co-expression network of LUM was determined by LinkedOmics, which showed that LUM expression was closely related to immune escape and the miR200 family. Furthermore, we studied the co-expression network of LUM and found that LUM could promote tumor metastasis and invasion. The Tumor Immune Estimation Resource website showed that LUM was closely related to immune infiltration and correlated with regulatory T cells, tumour-associated macrophages, and dendritic cells. We found that LUM cultivated cancer progression by targeting the miR200 family to promote epithelial-to-mesenchymal transition. These findings suggest that LUM is a potential target for inhibiting immune escape and carcinogenic pathways.

Endocan is a circulating proteoglycan, involved in immunity, inflammation, and endothelial function. It has been recently suggested as a biomarker of inflammation, increased angiogenesis, and cancer. In vitro studies have shown that endocan expression could be upregulated by inflammatory cytokines and proangiogenic molecules. High endocan levels were also shown in arthritic joint tissues and particularly in sites characterized by severe inflammation. This study was performed to evaluate endocan expression in chondrocytes stimulated with IL-ß. mRNA and related protein production were measured for endocan, TNF-α, and IL-6. NF-kB activity was also evaluated. IL-1ß treatment induced a significant upregulation of both endocan and the inflammatory parameters as well as NF-kB activity. The treatment of chondrocytes with the specific NF-kB inhibitor before IL-1ß stimulation was able to reduce endocan and the inflammatory markers over-expression. The results of our study indicated that endocan is also expressed in human chondrocytes; furthermore, consistent with previous results in other cell types and tissues, IL-1ß-induced inflammatory response involves the expression of endocan through NF-kB activation. In this context, endocan seems to be an important inflammatory marker associated with the activation of NF-kB pathway.

Perineuronal nets (PNNs) are an extracellular matrix structure rich in chondroitin sulfate proteoglycans (CSPGs), which preferentially encase parvalbumin-containing (PV+) interneurons. PNNs restrict cortical network plasticity but the molecular mechanisms involved are unclear. We found that reactivation of ocular dominance plasticity in the adult visual cortex induced by chondroitinase ABC (chABC)-mediated PNN removal requires intact signaling by the neurotrophin receptor TRKB in PV+neurons. Additionally, we demonstrate that chABC increases TRKB phosphorylation (pTRKB), while PNN component aggrecan attenuates brain-derived neurotrophic factor (BDNF)-induced pTRKB in cortical neurons in culture. We further found that protein tyrosine phosphatase σ (PTPσ, PTPRS), receptor for CSPGs, interacts with TRKB and restricts TRKB phosphorylation. PTPσ deletion increases phosphorylation of TRKBin vitroandin vivoin male and female mice, and juvenile-like plasticity is retained in the visual cortex of adult PTPσ-deficient mice (PTPσ+/−). The antidepressant drug fluoxetine, which is known to promote TRKB phosphorylation and reopen critical period-like plasticity in the adult brain, disrupts the interaction between TRKB and PTPσ by binding to the transmembrane domain of TRKB. We propose that both chABC and fluoxetine reopen critical period-like plasticity in the adult visual cortex by promoting TRKB signaling in PV+neurons through inhibition of TRKB dephosphorylation by the PTPσ-CSPG complex.SIGNIFICANCE STATEMENTCritical period-like plasticity can be reactivated in the adult visual cortex through disruption of perineuronal nets (PNNs) by chondroitinase treatment, or by chronic antidepressant treatment. We now show that the effects of both chondroitinase and fluoxetine are mediated by the neurotrophin receptor TRKB in parvalbumin-containing (PV+) interneurons. We found that chondroitinase-induced visual cortical plasticity is dependent on TRKB in PV+neurons. Protein tyrosine phosphatase σ (PTPσ, PTPRS), a receptor for PNNs, interacts with TRKB and inhibits its phosphorylation, and chondroitinase treatment or deletion of PTPσ increases TRKB phosphorylation. Antidepressant fluoxetine disrupts the interaction between TRKB and PTPσ, thereby increasing TRKB phosphorylation. Thus, juvenile-like plasticity induced by both chondroitinase and antidepressant treatment is mediated by TRKB activation in PV+interneurons.

Background To assess the effects of proteoglycan-depleted regions of annular disruptions on nerve ingrowth in the injury site in vivo. Methods New Zealand white rabbits (n = 18) received annular injuries at L3/4, L4/5, and L5/6. The experimental discs were randomly assigned to four groups: (a) an annular defect was created; (b) an annular defect implanted with a poly lactic-co-glycolic acid (PLGA)/fibrin/PBS plug; (c) an annular defect implanted with a PLGA/fibrin/chondroitinase ABC (chABC) plug; and (d) an uninjured L2/3 disc (control). Disc degeneration was evaluated by radiography, MRI, histology, and analysis of the proteoglycan (PG) content. Immunohistochemical detection of nerve fibers and chondroitin sulfate (CS) was performed. Results The injured discs produced progressive and reliable disc degeneration. In the defective discs, the lamellated appearance of AF (Annulus fibrosus) was replaced by extensive fibrocartilaginous-like tissue formation outside the injured sites. In contrast, newly formed tissue was distributed along small fissures, and small blood vessels appeared in the outer part of the disrupted area in the PLGA/fibrin/PBS discs. More sprouting nerve fibers grew further into the depleted annulus regions in the PLGA/fibrin/chABC discs than in the control discs and those receiving PLGA/fibrin/PBS. In addition, the innervation scores of the PLGA/fibrin/chABC discs were significantly increased compared with those of the PLGA/fibrin/PBS discs and defected discs. Conclusion ChABC-based PLGA/fibrin gel showed promising results by achieving biointegration with native annulus tissue and providing a local source for the sustained release of active chABC. Disc-derived PG-mediated inhibition of nerve and blood vessel ingrowth was abrogated by chABC enzymatic deglycosylation in an annular-injured rabbit disc degeneration model.

Cartilage tissue engineering is a promising option for repairing cartilage defects, although harvesting a large number of seeding cells remains a major challenge. Cartilage stem/progenitor cells (CSPCs) seem to be a promising cell source. Hypoxic extracellular vesicles (EVs) may play a major role in cell-cell and tissue-tissue communication. In the current study, we aimed to evaluate the effect of hypoxic adipose-derived stem cells (ADSCs)-derived EVs on CSPCs proliferation and differentiation. The characteristics of ADSCs-derived EVs were identified, and proliferation, migration, and cartilage-related gene expression of CSPCs were measured with or without the presence of hypoxic ADSCs-derived EVs. SEM, histological staining, biochemical and biomechanical analysis was performed to evaluate the effect of hypoxic ADSCs-derived EVs on CSPCs in alginate hydrogel culture. The results indicated that the majority of ADSC-derived EVs exhibited a round-shaped or cup-shaped morphology with a diameter of 40–1000 nm and expressed CD9, CD63, and CD81. CSPCs migration and proliferation were enhanced by hypoxic ADSCs-derived EVs, which also increased the expression of cartilage-related genes. The hypoxic ADSCs-derived EVs induce CSPCs to produce significantly more cartilage matrix and proteoglycan. In conclusion, hypoxic ADSCs-derived EVs improved the proliferation and chondrogenic differentiation of CSPCs for cartilage tissue engineering.

Systemic application of glucocorticoids is an essential anti-inflammatory and immune-modulating therapy for severe inflammatory or autoimmunity conditions. However, its long-term effects on articular cartilage of patients' health need to be further investigated. In this study, we studied the effects of dexamethasone (Dex) on the homeostasis of articular cartilage and the progress of destabilization of medial meniscus (DMM)-induced osteoarthritis (OA) in adult mice. Long-term administration of Dex aggravates the proteoglycan loss of articular cartilage and drastically accelerates cartilage degeneration under surgically induced OA conditions. In addition, Dex increases calcium content in calcified cartilage layer of mice and the samples from OA patients with a history of long-term Dex treatment. Moreover, long term usage of Dex results in decrease subchondral bone mass and bone density. Further studies showed that Dex leads to calcification of extracellular matrix of chondrocytes partially through activation of AKT, as well as promotes apoptosis of chondrocytes in calcified cartilage layer. Besides, Dex weakens the stress-response autophagy with the passage of time. Taken together, our data indicate that long-term application of Dex may predispose patients to OA and or even accelerate the OA disease progression development of OA patients.

Background: Asporin (ASPN), a member of the proteoglycan family, has been shown to have a close correlation with cancer progression. It is not known whether ASPN is an oncogenic driver or a tumor suppressor in human gastric cancer. We sought herein to determine the relationship between ASPN expression and clinicopathological features of gastric cancer. Patients and Methods: A total of 296 gastric cancer patients (diffuse type, n = 144; intestinal type, n = 152) were enrolled. The ASPN expression level in each case was analyzed by immunohistochemistry. Results: ASPN was mainly found on stromal cells, especially on fibroblasts in tumor stroma, i.e., cancer-associated fibroblasts. The ASPN expression on either cancer cells or stromal cells was significantly high in macroscopic scirrhous-type tumors (p < 0.001) and histologically abundant stroma-type tumors (p < 0.001). Interestingly, a Kaplan-Meier survival curve of the 144 cases of diffuse-type gastric cancer revealed a significantly poorer prognosis in patients with ASPN-positive expression (p = 0.043; log rank) compared to those with ASPN-negative expression, but the prognoses were not significantly different in these subgroups of the 152 cases of intestinal-type gastric cancer. A multivariate analysis with respect to overall survival showed that ASPN expression on stromal cells and/or cancer cells was significantly correlated with overall survival in patients with diffuse-type gastric cancer (p = 0.041). Conclusion: In gastric cancer, ASPN was expressed mainly on stromal cells and partially on cancer cells. ASPN expression on stromal cells and/or cancer cells might be a useful prognostic marker in patients with diffuse-type gastric cancer.

Recent in vitro evidence shows that glycosaminoglycans (GAGs) and proteoglycans (PGs) in bone matrix may functionally be involved in the tissue-level toughness of bone. In this study, we showed the effect of biglycan (Bgn), a small leucine-rich proteoglycan enriched in extracellular matrix of bone and the associated GAG subtype, chondroitin sulfate (CS), on the toughness of bone in vivo, using wild-type (WT) and Bgn deficient mice. The amount of total GAGs and CS in the mineralized compartment of Bgn KO mouse bone matrix decreased significantly, associated with the reduction of the toughness of bone, in comparison with those of WT mice. However, such differences between WT and Bgn KO mice diminished once the bound water was removed from bone matrix. In addition, CS was identified as the major subtype in bone matrix. We then supplemented CS to both WT and Bgn KO mice to test whether supplemental GAGs could improve the tissue-level toughness of bone. After intradermal administration of CS, the toughness of WT bone was greatly improved, with the GAGs and bound water amount in the bone matrix increased, while such improvement was not observed in Bgn KO mice or with supplementation of dermatan sulfate (DS). Moreover, CS supplemented WT mice exhibited higher bone mineral density and reduced osteoclastogenesis. Interestingly, Bgn KO bone did not show such differences irrespective of the intradermal administration of CS. In summary, the results of this study suggest that Bgn and CS in bone matrix play a pivotal role in imparting the toughness to bone most likely via retaining bound water in bone matrix. Moreover, supplementation of CS improves the toughness of bone in mouse models.

Aim: To observe the combined effect of platelet-rich plasma (PRP) and preconditioned adipose-derived mesenchymal stem cells (ADMSCs) on the injured articular cartilage of the rat. Materials & methods: Animals in the study received an intra-articular injection of PRP and preconditioned ADMSCs, both in combination and separately. The response to therapeutic intervention was evaluated by inflammatory markers, proteoglycans content, chondrogenesis and gene expression analyses. Results: The combined therapy resulted in a reduction of IL-6 and TNF-α, increased proteoglycan content of the articular cartilage, upregulation of Acan, Col2a1 and PCNA genes. Downregulation of Col1a1, Col10a1 and Casp3 genes was observed as compared with the untreated osteoarthritis rat model. Conclusion: PRP potentiates the effects of ADMSCs on the repair of damaged articular cartilage.

The resveratrol-enriched transgenic rice line Iksan526 (IS526), first developed by the Rural Development Administration of Korea using genetic engineering techniques, shows beneficial health effects in mitigating metabolic syndrome and obesity. However, the effects of IS526 on the differentiation of chondrocytes and the underlying mechanism have not been investigated in detail. In this study, the effects and cellular regulatory mechanisms of IS526 on rabbit articular chondrocytes were examined. Following IS526 callus extract treatment, the expression levels of differentiation-related proteins were detected via western blotting, Alcian blue staining and immune-luorescence staining. IS526 decreased the type II collagen and proteoglycan levels in dose- and time-dependent manners. We further analyzed the effects of IS526 on skeleton genesis in zebrafish larvae using Alcian blue staining, which showed a reduction in cartilage formation along with increased production of matrix metalloproteinase (MMP)-13. IS526 also increased the phosphorylation of ERK1/2 and p38 kinase but inhibited the phosphorylation of Akt. Pharmacological inhibition of MMP-13 blocked the IS526-induced decrease in type II collagen levels. Inhibition of p38 kinase or PI-3K/Akt with SB203580 and LY294002 enhanced the suppression of type II collagen, but the blockage of ERK-1/2 by PD98059 rescued IS526-induced dedifferentiation. These results suggested that IS526 regulates type II collagen and MMP-13 expression via the ERK1/2 and PI-3K/Akt pathways in rabbit articular chondrocytes.

Many factors contribute to the development and progression of intervertebral disc (IVD) degeneration. This study was designed to assess the effects of compressive load magnitude on IVD metabolism. It was hypothesized that as load magnitude increased, there would be a significant increase in release of proinflammatory and degradative biomarkers, and a significant decrease in tissue proteoglycan (GAG) and collagen contents compared with unloaded controls. IVD whole organ functional spinal units (FSU) consisting of cranial and caudal body halves, cartilage endplates, and IVD (n = 36) were harvested from the tails of six Sprague Dawley rats, and FSUs were cultured at 0.0 MPa, 0.5 MPa, or 1.0 MPa at 0.5 Hz for 3 days. After culture, media were collected for biomarker analysis and FSUs were analyzed for extracellular matrix composition. Significant differences were determined using a one-way analysis of variance or Kruskal-Wallis test and post hoc analyses. Media concentrations of IFN-γ, IL-6, IL-1β, and MMP-8 were significantly higher in the 0.5 MPa compared with the 0.0 MPa group. Media concentrations of PGE2 and TIMP-1 were significantly higher in the 1.0 MPa group compared with the 0.0 MPa group, and media PGE2 was significantly higher in the 1.0 MPa group compared with the 0.5 MPa group. Media GAG content was significantly higher in the 1.0 MPa group compared with the 0.0 MPa group, and percent GAG in the tissue was significantly lower in 0.5 MPa and 1.0 MPa groups compared with the 0.0 MPa group. Clinical Significance: These data suggest that there are magnitude-dependent inflammatory and degradative IVD responses to cyclic loading, which may contribute to IVD degeneration.

Proposed mechanisms for lumican in joint contracture include: (a) increased lumican with posttraumatic damage that has caused a transition from a normal joint capsule to joint contracture; and (b) stimulating synovial fibroblasts with recombinant lumican leads to increased transcription of alpha‐smooth muscle actin, matrix metallopeptidase 9, Collagen I, plasminogen activator inhibitor 1 and transforming growth factor‐β in vitro, possibly through the transforming growth factor‐β pathway. Thus, lumican may affect capsule fibrosis in joint contracture., Joint contracture (also known as arthrofibrosis) is a fibrotic joint disorder characterized by excessive collagen production to form fibrotic scar tissue and adhesions within joint capsules. This can severely affect day‐to‐day activities and quality of life because of a restricted range of motion in affected joints. The precise pathogenic mechanism underlying joint contractures is not fully understood. Lumican belongs to the class II small leucine‐rich repeat proteoglycan superfamily, which makes up collagen fibrils in the extracellular matrix. Lumican is ubiquitously expressed in the skin, liver, heart, uterus and articular cartilage and has reported roles in cell migration, proliferation, angiogenesis and Toll‐like receptor 4 signaling. Previous research has suggested that lumican is involved in the pathogenesis of several fibrotic diseases. Because joint contracture resembles a fibrotic disease, we aimed to investigate the role of lumican in the development of joint contracture in vitro. Here, we showed that protein levels were up‐regulated in the fibrotic joint capsule versus control. We observed that lumican significantly enhanced the proliferation, migration and fibroblast–myofibroblast transition of synovial fibroblasts. Moreover, lumican led to increased transcription of alpha‐smooth muscle actin, matrix metallopeptidase 9, Collagen I, plasminogen activator inhibitor 1 and transforming growth factor‐β in vitro. Lumican treatment promoted collagen lattice contraction in a dose‐dependent manner as early as 24 h after treatment. Thus, our studies reveal that lumican could promote fibroblast–myofibroblast transition and joint contracture.

Hyaluronan and proteoglycan link protein 1 (HAPLN1) stabilizes interactions between two important extracellular matrix (ECM) macromolecules, versican and hyaluronan, which facilitate proliferation of fibroblasts and their conversion to myofibroblasts. However, the role of HAPLN1 in these events has not been studied. Using immunocytochemistry, cellular and ECM locations of HAPLN1 were evaluated in cultured human lung fibroblasts during proliferation and conversion to myofibroblasts. HAPLN1 localized to pericellular matrices, associating with both versican and hyaluronan in the ECM and on the cell surface. Nuclear and total HAPLN1 immunostaining increased after myofibroblast induction. Confocal microscopy showed HAPLN1 predominant in the ECM under cells while versican predominated above cells. Versican and HAPLN1 were also juxtaposed in columnar inclusions in the cytoplasm and nucleus. Nuclear HAPLN1 staining in interphase cells redistributed to the cytosol during mitosis. In the absence of TGF-β1, addition of exogenous bovine HAPLN1 (together with aggrecan G1) facilitated myofibroblast formation, as seen by significant upregulation of α-smooth muscle actin (SMA) staining, while adding full-length bovine versican had no effect. Increased compaction of hyaluronan-rich ECM suggests that HAPLN1 plus G1 addition affects hyaluronan networks and myofibroblast formation. These observations demonstrate changes in both extracellular and intracellular localization of HAPLN1 during fibroblast proliferation and myofibroblast conversion suggesting a possible role in fibrotic remodeling

Low back pain is a global health problem that is frequently caused by intervertebral disc degeneration (IVDD). Sulfated glycosaminoglycans (sGAGs) give the healthy nucleus pulposus (NP) a high fixed charge density (FCD), which creates an osmotic pressure that enables the disc to withstand high compressive forces. However, during IVDD sGAG reduction in the NP compromises biomechanical function. The aim of this study was to develop an ex vivo NP explant model with reduced sGAG content and subsequently investigate biomechanical restoration via injection of proteoglycan-containing notochordal cell-derived matrix (NCM). Bovine coccygeal NP explants were cultured in a bioreactor chamber and sGAG loss was induced by chondroitinase ABC (chABC) and cultured for up to 14 days. Afterwards, diurnal loading was studied, and explant restoration was investigated via injection of NCM. Explants were analyzed via histology, biochemistry, and biomechanical testing via stress relaxation tests and height measurements. ChABC injection induced dose-dependent sGAG reduction on Day 3, however, no dosing effects were detected after 7 and 14 days. Diurnal loading reduced sGAG loss after injection of chABC. NCM did not show an instant biomechanical (equilibrium pressure) or biochemical (FCD) restoration, as the injected fixed charges leached into the medium, however, NCM stimulated proliferation and increased Alcian blue staining intensity and matrix organization. NCM has biological repair potential and biomaterial/NCM combinations, which could better entrap NCM within the NP tissue, should be investigated in future studies. Concluding, chABC induced progressive, time-, dose- and loading-dependent sGAG reduction that led to a loss of biomechanical function. Keywords. biomechanics | intervertebral disc | matrix degradation | low back pain | proteoglycans.

In this paper we characterize the function of Xylosyltransferase 2 (XylT2) in different tissues to investigate the role XylT2 has in the proteoglycan (PG) biochemistry of multiple organs. The results show that in all organs examined there is a widespread and significant decrease in total XylT activity in Xylt2 knock out mice (Xylt2−/−). This decrease results in increased organ weight differences in lung, heart, and spleen. These findings, in addition to our previous findings of increased liver and kidney weight with loss of serum XylT activity, suggest systemic changes in organ function due to loss of XylT2 activity. The Xylt2−/− mice have splenomegaly due to enlargement of the red pulp area and enhanced pulmonary response to bacterial liposaccharide. Tissue glycosaminoglycan composition changes are also found. These results demonstrate a role of XylT2 activity in multiple organs and their PG content. Because the residual XylT activity in the Xylt2−/− is due to xylosyltransferase 1 (XylT1), these studies indicate that both XylT1 and XylT2 have important roles in PG biosynthesis and organ homeostasis.

Advances in reagents, methodologies, analytic platforms, and tools have resulted in a dramatic transformation of the research pathology laboratory. These advances have increased our ability to efficiently generate substantial volumes of data on the expression and accumulation of mRNA, proteins, carbohydrates, signaling pathways, cells, and structures in healthy and diseased tissues that are objective, quantitative, reproducible, and suitable for statistical analysis. The goal of this review is to identify and present how to acquire the critical information required to measure changes in tissues. Included is a brief overview of two morphometric techniques, image analysis and stereology, and the use of artificial intelligence to classify cells and identify hidden patterns and relationships in digital images. In addition, we explore the importance of preanalytical factors in generating high-quality data. This review focuses on techniques we have used to measure proteoglycans, glycosaminoglycans, and immune cells in tissues using immunohistochemistry and in situ hybridization to demonstrate the various morphometric techniques. When performed correctly, quantitative digital pathology is a powerful tool that provides unbiased quantitative data that are difficult to obtain with other methods.