Proteoglycan Research Articles

Effects of Glucose Deprivation on ATP and Proteoglycan Production of Intervertebral Disc Cells under Hypoxia

As the most common cause of low back pain, the cascade of intervertebral disc (IVD) degeneration is initiated by the disappearance of notochordal cells and progressive loss of proteoglycan (PG). Limited nutrient supply in the avascular disc environment restricts the production of ATP which is an essential energy source for cell survival and function such as PG biosynthesis. The objective of this study was to examine ATP level and PG production of porcine IVD cells under prolonged exposure to hypoxia with physiological glucose concentrations. The results showed notochordal NP and AF cells responded differently to changes of oxygen and glucose. Metabolic activities (including PG production) of IVD cells are restricted under the in-vivo nutrient conditions while NP notochordal cells are likely to be more vulnerable to reduced nutrition supply. Moreover, provision of energy, together or not with genetic regulation, may govern PG production in the IVD under restricted nutrient supply. Therefore, maintaining essential levels of nutrients may reduce the loss of notochordal cells and PG in the IVD. This study provides a new insight into the metabolism of IVD cells under nutrient deprivation and the information for developing treatment strategies for disc degeneration.

Metabolism of tumor cell in tumor micro environment assist immune escape.

e15181 Background: Tumor cell’s metabolism has been identified assisting immune evasion in tumor micro environment (TME), and it is very different from that of immune cells. Previous studies mostly focus on bulk or cell lines transcriptome; we aim to find the specific metabolic model of tumor cell based on vast single cell RNA-seq data. Methods: Public transcriptome of 7186 cells (SMART-Seq2) from 33 melanoma tumors in Benjamin’s study were collected from Single Cell Portal of Broad Institute, and metabolic genes were obtained from Human Metabolome Database (HMDB). Raw count data was preprocessed and analyzed using Seurat R package. Statistical analysis and data visualization were all carried out using R software. Results: Metabolic signature genes of malignant cells (Mals), CD8+ T cells, CD4+ T cells, B cells, natural killer (NK) cells, macrophages, cancer associated fibroblasts (CAFs), and endothelial cells (Endos) were extracted using Seurat. Mals, CAFs, Endos and macrophages shared similar metabolism patterns (high-level glycolysis, pyruvate metabolism, purine metabolism and specific proteoglycans profile compared to immune cells, p < 0.01). Glutathione metabolism activity in Mal was higher than that in immune cell, especially GPX1, GPX4 and GSTO1 genes were highly expressed in Mal ( fc > 2, p < 0.01). Compared to immune cells, genes participate in glycolysis, PFKL, ADH5, ENO2, G6PC3, PGM1, ALDH1B1, PFKM, ALDH7A1, GAPDHS, TPI1, PGK1, LDHA, GPI, PKM, LDHB, ENO1, ALDOA and GAPDH were highly expressed in Mals, CAFs and Endos ( fc > 2, p < 0.01). Noteworthily, LGALS1, an inducer of T-cell apoptosis, was highly expressed in Mals and CAF ( fc > 2, p < 0.01). Otherwise, proteoglycans (PGs) in cancer, which contribute to cancer pathogenesis, exhibited high variation from immune cells. Conclusions: We firstly highlight the differential metabolism between tumor and immune cells in TME. Mals, CAFs, endothelial cells and macrophages can assist in evading immune surveillance through reshaping their metabolic patterns at cell level. This study is helpful for exploring new target of metabolic based anti-cancer immunotherapy.

A viscoelastic two-dimensional network model of the lung extracellular matrix.

The extracellular matrix (ECM) comprises a large proportion of the lung parenchymal tissue and is an important contributor to the mechanical properties of the lung. The lung tissue is a biologically active scaffold with a complex ECM matrix structure and composition that provides physical support to the surrounding cells. Nearly all respiratory pathologies result in changes in the structure and composition of the ECM; however, the impact of these alterations on the mechanical properties of the tissue is not well understood. In this study, a novel network model was developed to incorporate the combinatorial effect of lung tissue ECM constituents such as collagen, elastin and proteoglycans (PGs) and used to mimic the experimentally derived length-tension response of the tissue to uniaxial loading. By modelling the effect of collagen elasticity as an exponential function with strain, and in concert with the linear elastic response of elastin, the network model's mechanical response matched experimental stress-strain curves from the literature. In addition, by incorporating spring-dashpot viscoelastic elements, to represent the PGs, the hysteresis response was also simulated. Finally, by selectively reducing volume fractions of the different ECM constituents, we were able to gain insight into their relative mechanical contribution to the larger scale tissue mechanical response.

Label-Free Infrared Spectral Histology of Skin Tissue Part I: Impact of Lumican on Extracellular Matrix Integrity.

Proteoglycans (PG) play an important role in maintaining the extracellular matrix (ECM) integrity. Lumican, a small leucine rich PG, is one such actor capable of regulating such properties. In this study, the integrity of the dermis of lumican-deleted Lum–/– vs. wild-type mice was investigated by conventional histology and by infrared spectral histology (IRSH). Infrared spectroscopy is a non-invasive, rapid, label-free and sensitive technique that allows to probe molecular vibrations of biomolecules present in a tissue. Our IRSH results obtained on control (WT, n = 3) and Lum–/– (n = 3) mice showed that different histological structures were identified by using K-means clustering and validated by hematoxylin eosin saffron (HES) staining. Furthermore, an important increase of the dermis thickness was observed in Lum–/– compared to WT mice. In terms of structural information, analysis of the spectral images also revealed an intra-group homogeneity and inter-group heterogeneity. In addition, type I collagen contribution was evaluated by HES and picrosirius red staining as well as with IRSH. Both techniques showed a strong remodeling of the ECM in Lum–/– mice due to the looseness of collagen fibers in the increased dermis space. These results confirmed the impact of lumican on the ECM integrity. The loss of collagen fibers organization due to the absence of lumican can potentially increase the accessibility of anti-cancer drugs to the tumor. These results are qualitatively interesting and would need further structural characterization of type I collagen fibers in terms of size, organization, and orientation.

Matrilin-3 alleviates extracellular matrix degradation of nucleus pulposus cells via induction of IL-1 receptor antagonist.

The intervertebral disc contains abundant extracellular matrix (ECM) imbued with proteoglycans, collagens, and water. With the development of intervertebral disc degeneration (IVDD), the ECM undergoes changes characterized by loss of water content, proteoglycans, and collagen content. The purpose of this study was to explore the vital role of Matrilin-3, an ECM protein involved in the progress of IVDD. NP cells were isolated from the patients' disc samples and exposed to recombinant human (rh)-Matrilin-3 protein (MATN3), and IL-1β is used as a reducer of nucleus pulposus (NP) cells degeneration. Matrilin-3 and IL-1 receptor antagonist (IL-1Ra) were knocked down by siRNA transfection. Messenger RNA expressions of IL-1Ra, Collagen II, aggrecan, MMP-13, and ADAMTS-5 were determined using Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). Later, the protein levels of IL-Ra, Collagen II, and aggrecan were also detected by Western blot. The IL-1Ra, MMP-13, and ADAMTS-5 dose of the supernatants in the culture medium was determined by enzyme linked immunosorbent assay (ELISA). Finally, immunofluorescence was used to expose the expression of Collagen II, aggrecan, and Collagen X. It was found that the expression of IL-1Ra was markedly increased in the present of MATN3 or IL-1β, especially these two at once. Besides, MATN3 could upregulate Collagen II and aggrecan expressions, as well as inhibit the MMP-13 and Collagen X production of NP cells. However, the protective effects of Collagen II and aggrecan were abolished after Matrilin-3 silenced. Furthermore, IL-1β downregulated the Collagen II and aggrecan but promoted the MMP-13 and Collagen X levels of NP cells, which were antagonized by the action of MATN3. Surprisingly, silencing of IL-1Ra significantly abolished the MATN3-induced the protective effects of ECM in NP cells. This study provides a novel viewpoint of Matrilin-3 in the ECM stability of NP due to its ability to activate IL-1Ra. It is considered that MATN3 efficiently protects ECM degeneration of human NP cells related to maintain the content of Collagen II and aggrecan, as well as inflammatory inhibition.

Pharmacological activation of protein tyrosine phosphatase SHP-1 ameliorates autoimmune arthritis in mice

Abstract Background The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is known to exert negative regulatory effects on immune cell signaling. We observed previously that mice overexpressing an SHP-1 transgene were resistant to the development of autoimmune arthritis and showed decreased immune responses in the cartilage proteoglycan (PG)-induced (PGIA) model of rheumatoid arthritis (RA). We sought to explore if pharmacological activation of SHP-1 in wildtype (WT) mice prevents or ameliorates arthritis. Methods To induce PGIA, mice were immunized with human (PG) in adjuvant. For in vivo SHP-1 activation, mice were treated with regorafenib (BAY 73-4506) per os either before arthritis symptoms developed (preventive treatment) or shortly after the initial signs of arthritis (therapeutic treatment). Regorafenib administration was continued for at least 21 days while arthritis incidence and severity were monitored. Results Preventive regorafenib treatment of WT mice significantly decreased arthritis severity (10 or 20 mg/kg) and incidence compared to vehicle treatment. Preventive treatment (10 mg/kg) increased tyrosine phosphatase activity of spleen cells and decreased IFNγ secretion by splenic T cells in vitro. Therapeutic treatment with 15 mg/kg decreased the severity of arthritis and lowered IFNγ production. Conclusion Our studies are the first to investigate the role of SHP-1 in autoimmune arthritis. We found that pharmacological activation of SHP-1 in vivo prevented the development of autoimmune arthritis or ameliorated arthritis severity when applied after the disease onset in the PGIA model of RA. Activation of SHP-1 might open a new therapeutic perspective in RA treatment.

Proteoglycan-Dependent Endo-Lysosomal Fusion Affects Intracellular Survival of Salmonella Typhimurium in Epithelial Cells.

Proteoglycans (PGs) are glycoconjugates which are predominately expressed on cell surfaces and consist of glycosaminoglycans (GAGs) linked to a core protein. An initial step of GAGs assembly is governed by the β-D-xylosyltransferase enzymes encoded in mammals by the XylT1/XylT2 genes. PGs are essential for the interaction of a cell with other cells as well as with the extracellular matrix. A number of studies highlighted a role of PGs in bacterial adhesion, invasion, and immune response. In this work, we investigated a role of PGs in Salmonella enterica serovar Typhimurium (S. Typhimurium) infection of epithelial cells. Gentamicin protection and chloroquine resistance assays were applied to assess invasion and replication of S. Typhimurium in wild-type and xylosyltransferase-deficient (ΔXylT2) Chinese hamster ovary (CHO) cells lacking PGs. We found that S. Typhimurium adheres to and invades CHO WT and CHO ΔXylT2 cells at comparable levels. However, 24 h after infection, proteoglycan-deficient CHO ΔXylT2 cells are significantly less colonized by S. Typhimurium compared to CHO WT cells. This proteoglycan-dependent phenotype could be rescued by addition of PGs to the cell culture medium, as well as by complementation of the XylT2 gene. Chloroquine resistance assay and immunostaining revealed that in the absence of PGs, significantly less bacteria are associated with Salmonella-containing vacuoles (SCVs) due to a re-distribution of endocytosed gentamicin. Inhibition of endo-lysosomal fusion by a specific inhibitor of phosphatidylinositol phosphate kinase PIKfyve significantly increased S. Typhimurium burden in CHO ΔXylT2 cells demonstrating an important role of PGs for PIKfyve dependent vesicle fusion which is modulated by Salmonella to establish infection. Overall, our results demonstrate that PGs influence survival of intracellular Salmonella in epithelial cells via modulation of PIKfyve-dependent endo-lysosomal fusion.

Structural Insights Into How Proteoglycans Determine Chemokine-CXCR1/CXCR2 Interactions: Progress and Challenges

Proteoglycans (PGs), present in diverse environments, such as the cell membrane surface, extracellular milieu, and intracellular granules, are fundamental to life. Sulfated glycosaminoglycans (GAGs) are covalently attached to the core protein of proteoglycans. PGs are complex structures, and are diverse in terms of amino acid sequence, size, shape, and in the nature and number of attached GAG chains, and this diversity is further compounded by the phenomenal diversity in GAG structures. Chemokines play vital roles in human pathophysiology, from combating infection and cancer to leukocyte trafficking, immune surveillance, and neurobiology. Chemokines mediate their function by activating receptors that belong to the GPCR class, and receptor interactions are regulated by how, when, and where chemokines bind GAGs. GAGs fine-tune chemokine function by regulating monomer/dimer levels and chemotactic/haptotactic gradients, which are also coupled to how they are presented to their receptors. Despite their small size and similar structures, chemokines show a range of GAG-binding geometries, affinities, and specificities, indicating that chemokines have evolved to exploit the repertoire of chemical and structural features of GAGs. In this review, we summarize the current status of research on how GAG interactions regulate ELR-chemokine activation of CXCR1 and CXCR2 receptors, and discuss knowledge gaps that must be overcome to establish causal relationships governing the impact of GAG interactions on chemokine function in human health and disease.

Glycosylation of DMP1 promotes bone reconstruction in long bone defects

The regeneration of bone defects is necessary for the successful healing. During the process of healing, callus plays crucial roles in providing the stable bone-reconstruction environment. The callus is consisted of various large molecules including collagen proteins, non-collagen proteins and proteoglycans (PGs), which are involved in maintaining mechanical strength and interacting with cytokines and grow factors in the injury sites. Recently, our data have found that the PG form of Dentin Matrix Protein 1 (DMP1-PG), which is a newly identified PG, was richly expressed in the bone defect sites. Previous researches have demonstrated the special role of DMP1-PG in chondrogenesis and endochondral ossification, however, the knowledge about the role of DMP1-PG in bone defect repair is still limited. To further detect the potential function of DMP1-PG in the defect healing, we employed a bone defect intramembranous ossification model using the glycosylation site mutant DMP1-PG (S89-G89, S89G-DMP1) mouse. The morphologic changes of calluses and abnormal expression levels of osteogenesis genes were displayed in the injury sites in S89G-DMP1 mice. In addition, impaired BMP-Smad signaling pathway was observed due to the deficiency of DMP1-PG. Collectively, our findings indicated that the DMP1-PG is one of key proteoglycans in the process of defect healing via regulating the osteogenesis.

Alterations in ECM signature underscore multiple sub-phenotypes of intervertebral disc degeneration

The intervertebral disc is a specialized connective tissue critical for absorption of mechanical loads and providing flexibility to the spinal column. The disc ECM is complex and plays a vital role in imparting tissue its biomechanical function. The central NP is primarily composed of large aggregating proteoglycans (PGs) while surrounding AF is composed of fibrillar collagens, I and II. Aggrecan and versican in particular, due to their high concentration of sulfated GAG chains form large aggregates with hyaluronic acid (HA) and provide water binding capacity to the disc. Degradation of aggrecan core protein due to aggrecanase and MMP activity, SNPs that affect number of chondroitin sulfate (CS) substitutions and alteration in enzymes critical in synthesis of CS chains can impair the aggrecan functionality. Similarly, levels of many matrix and matrix-related molecules e.g. Col2, Col9, HAS2, ccn2 are dysregulated during disc degeneration and genetic animal models have helped establish causative link between their expression and disc health. In the degenerating and herniated discs, increased levels of inflammatory cytokines such as TNF-α, IL-1β and IL-6 are shown to promote matrix degradation through regulating expression and activity of critical proteases and stimulate immune cell activation. Recent studies of different mouse strains have better elucidated the broader impact of spontaneous degeneration on disc matrix homeostasis. SM/J mice showed an increased cell apoptosis, loss of cell phenotype, and cleavage of aggrecan during early stages followed by tissue fibrosis evident by enrichment of several collagens, SLRPs and fibronectin. In summary, while disc degeneration encompasses wide spectrum of degenerative phenotypes extensive matrix degradation and remodeling underscores all of them.

Skeletal Dysplasias Caused by Sulfation Defects.

Proteoglycans (PGs) are macromolecules present on the cell surface and in the extracellular matrix that confer specific mechanical, biochemical, and physical properties to tissues. Sulfate groups present on glycosaminoglycans, linear polysaccharide chains attached to PG core proteins, are fundamental for correct PG functions. Indeed, through the negative charge of sulfate groups, PGs interact with extracellular matrix molecules and bind growth factors regulating tissue structure and cell behavior. The maintenance of correct sulfate metabolism is important in tissue development and function, particularly in cartilage where PGs are fundamental and abundant components of the extracellular matrix. In chondrocytes, the main sulfate source is the extracellular space, then sulfate is taken up and activated in the cytosol to the universal sulfate donor to be used in sulfotransferase reactions. Alteration in each step of sulfate metabolism can affect macromolecular sulfation, leading to the onset of diseases that affect mainly cartilage and bone. This review presents a panoramic view of skeletal dysplasias caused by mutations in genes encoding for transporters or enzymes involved in macromolecular sulfation. Future research in this field will contribute to the understanding of the disease pathogenesis, allowing the development of targeted therapies aimed at alleviating, preventing, or modifying the disease progression.

Activin A-Mediated Regulation of XT-I in Human Skin Fibroblasts.

Fibrosis is a fundamental feature of systemic sclerosis (SSc) and is characterized by excessive accumulation of extracellular matrix components like proteoglycans (PG) or collagens in skin and internal organs. Serum analysis from SSc patients showed an increase in the enzyme activity of xylosyltransferase (XT), the initial enzyme in PG biosynthesis. There are two distinct XT isoforms—XT-I and XT-II—in humans, but until now only XT-I is associated with fibrotic remodelling for an unknown reason. The aim of this study was to identify new XT mediators and clarify the underlying mechanisms, in view of developing putative therapeutic anti-fibrotic interventions in the future. Therefore, we used different cytokines and growth factors, small molecule inhibitors as well as small interfering RNAs, and assessed the cellular XT activity and XYLT1 expression in primary human dermal fibroblasts by radiochemical activity assays and qRT-PCR. We identified a new function of activin A as a regulator of XYLT1 mRNA expression and XT activity. While the activin A-induced XT-I increase was found to be mediated by activin A receptor type 1B, MAPK and Smad pathways, the activin A treatment did not alter the XYLT2 expression. Furthermore, we observed a reciprocal regulation of XYLT1 and XYLT2 transcription after inhibition of the activin A pathway components. These results improve the understanding of the differential expression regulation of XYLT isoforms under pathological fibroproliferative conditions.

The Role of Proteoglycans in Glomerular Physiology and Pathophysiology

BackgroundDiabetic kidney disease (DKD) is the leading cause of renal failure in the world. Diabetes is associated with damage to the endothelial glycocalyx (eGCX), the layer of proteoglycans (PGs) and other negatively charged molecules covering the glomerular endothelial capillaries. The eGCX restrain the flow of charged molecules, such as albumin, over the filtration barrier in the kidney. Loss of the glomerular eGCX leads to proteinuria without other visible damage to the filtration barrier, but the detailed structure and composition is still largely unknown. The aim of this study was to gain new knowledge about the composition and role of the EGCX in health and DKD.MethodsThe negatively charged PGs in the eGCX was eluted from rat kidneys using 1 M NaCl solution (high salt, HS). In addition 1 M mannitol was used as osmotic control (HO) and 0.15 M NaCl to mimic physiological salt concentrations (NS). Solutions were introduced intra‐arterially to rat kidneys under anesthesia in vivo. Venous effluent was analyzed using mass spectrometry. Fractional clearance of albumin and GFR was measured. Electron microscopy (EM) was used to investigate morphological changes. Expression of PGs and PG related genes in glomeruli from patients with DKD was investigated using deep sequencing data from a Swedish DKD cohort (DKD, n=19, controls; pre‐transplant biopsies, n=20).ResultsWe identified 15 PGs in the eluate from rats. PGs were found in highest yields in the HS samples. EM demonstrated that the eGCX thickness was significantly reduced in the HS rats −28% (p<0.05) compared to NS. Rats perfused with HS had increased fractional clearance of albumin and reduced GFR, compared to NS and HO, 10 minutes after perfusion. In glomeruli from patients with DKD 12 PGs were found to be significantly regulated, and 4 of these PGs were also identified in the eGCX eluates from rats. There was an overall decrease in expression of enzymes responsible for building up PG side chains, as well as an increase in proteins involved in PG degradation.ConclusionThe eGCX itself represents a dynamic structure with significant molecular turnover. In our study, we identified several PGs novel to the glomerular eGCX. We show that loss of eGCX leads to proteinuria and reduced GFR, strongly supporting that the eGCX is important for preventing proteinuria. In glomeruli from patients with DKD we found significant alterations in the gene expression of PGs and the enzymes regulating their composition. Further investigation is needed to clarify in detail when and how these alterations appear in DKD.

Genome‐wide Screening Uncovers Transforming Growth Factor‐β3 as Regulator of Glycosaminoglycan Assembly

Heparan sulfate (HS) and chondroitin sulfate (CS) are glycosaminoglycans covalently linked to proteoglycans (PG) present on all cell surfaces and in the extracellular matrix. They play a complex role in carcinogenesis as they are important for extracellular matrix remodeling as well as cell‐cell and cell‐matrix interactions. These processes are altered during malignancy, when tumor cells invade and systemically spread in different tissues. We have developed a genome wide screening method using CRISPR‐Cas9 gene targeting to identify novel genes involved in glycosaminoglycan metabolism. The approach utilizes agents that bind specifically to cell surface HS. One of the top hits was transforming growth factor‐β3 (TGF‐β3), a growth factor that controls multiple genes with tumor‐suppressing and tumor‐promoting effects. Cell surface levels of PG were evaluated in A375 melanoma cells after treatment with soluble TGF‐β1 or TGF‐β3 in the presence or absence of a TGF‐β type I receptor inhibitor (SB‐431542) using specific agents that bind to glycosaminoglycans, including mAbs 10E4 or CS56, guanidinylated neomycin and fibroblast growth factor‐2 (FGF2). Both TGF‐β isoforms stimulated cell surface HSPG and CSPG expression, that was blocked by SB‐431542. Inactivation of TGF‐β3 expression by CRISPR/Cas9 had the opposite effect. These findings show that TGF‐β3 is a newly identified regulatory factor of PG assembly in melanoma cells and that tonic expression of TGF‐β3 regulates PG production in a cell autonomous manner. Further exploration how TGF‐β affects disorders associated with altered PG formation in cancer might lead to innovative solutions to alter tumor cell growth and metastasis.Support or Funding InformationWe acknowledge financial support by the DFG (research fellowship, project number 420160411) and the NIH (fellowship award, K12HL141956).

Approaches for In Situ Monitoring of Matrix Development in Hydrogel-Based Engineered Cartilage.

Near infrared (NIR) spectroscopy using a fiber optic probe shows great promise for the nondestructive in situ monitoring of tissue engineered construct development; however, the NIR evaluation of matrix components in samples with high water content is challenging, as water absorbances overwhelm the spectra. In this study, we established approaches by which NIR spectroscopy can be used to select optimal individual engineered hydrogel constructs based on matrix content and mechanical properties. NIR spectroscopy of dry standard compounds allowed identification of several absorbances related to collagen and/or proteoglycan (PG), of which only two could be identified in spectra obtained from hydrated constructs, at ∼5940 and 5800 cm-1. In dry sample mixtures, the ratio of these peaks correlated positively to collagen and negatively to PG. In NIR spectra from engineered cartilage hydrogels, these peaks reflected higher collagen and PG content and dynamic modulus values, permitting the differentiation of constructs with poor and good matrix development. Similarly, the increasing baseline offset in raw NIR spectra also reflected matrix development in hydrated constructs. However, weekly monitoring of NIR spectra and the peaks at ∼5940 and 5800 cm-1 was not adequate to differentiate individual constructs based on matrix composition. Interestingly, changes in the baseline offset of raw spectra could be used to evaluate the growth trajectory of individual constructs. These results demonstrate an optimal approach for the use of fiber optic NIR spectroscopy for in situ monitoring of the development of engineered cartilage, which will aid in identifying individual constructs for implantation. Impact statement A current demand in tissue engineering is the establishment of nondestructive approaches to evaluate construct development during growth in vitro. In this article, we demonstrate original nondestructive approaches by which fiber optic NIR spectroscopy can be used to assess matrix (PG and collagen) formation and mechanical properties in hydrogel-based constructs. Our data provide a cohesive molecular-based approach for in situ longitudinal evaluation of construct development during growth in vitro. The establishment of these approaches is a valuable step toward the real-time identification and selection of constructs with optimal properties, which may lead to successful tissue integration upon in vivo implantation.

Knee joint distraction-induced shift from catabolic to anabolic state occurs after the distraction period

Purpose: Knee joint distraction (KJD) is a validated joint-preserving treatment strategy for severe osteoarthritis (OA) that provides long-term clinical and structural improvement. Data from both human trials and animal models indicate clear cartilage regeneration from 6 months and onwards post-KJD. However, recent work showed that during distraction, the balance between catabolic and anabolic indicators is directed towards catabolism, as indicated by collagen type 2 markers, proteoglycan (PG) turnover and a catabolic transcription profile [unpublished data].

Reduced Proteoglycan-Rich Layer and Altered Immunotolerance Capacity of C4ST-1-Depleted Bone Marrow Mesenchymal Stem Cells on Titanium Dioxide

Successful osseointegration of a bone and implant directly must be achieved for treatment of a dental implant. However, the mechanism of osseointegration at the molecular level has yet to be fully elucidated. In this study, we focused on the proteoglycans (PGs)-rich layer between titanium dioxide (TiO2) and bone and chondroitin-4-sulfate transferase-1 (C4ST-1) that forms the sugar chain in PGs. Human bone marrow mesenchymal stem cells (hBMSCs) depleted of C4ST-1 were cultured on TiO2, and the interface between hBMSCs and TiO2 was analyzed by transmission electron microscopy. Further, the immunotolerance capacity, cell proliferation, initial attachment, and calcification were analyzed in vitro. At 14 days cultivation, a PGs-rich layer was observed between hBMSCs and TiO2. However, the PGs-rich layer was reduced in C4ST-1-depleted hBMSCs on TiO2. Further, real-time RT-PCR showed that conditioned media increased gene expression levels of M1-macrophage markers in human macrophages. However, C4ST-1 did not affect calcification, cell proliferation, and initial cell attachment on TiO2. These results suggested that C4ST-1 in hBMSCs affected the width of the PGs-rich layer on TiO2 and their immunotolerance capacity.

Morphologic Study on Lymphocyte Homing in Duck Tembusu Virus-Infected Duck Spleen.

The present study was designed to analyze the histologic and cytologic changes of lymphocyte homing in noninfected and duck Tembusu virus (DTMUV)-infected duck spleens. At first, we investigated the noninfected structure that facilitates lymphocyte homing. Under light and electron microscopy, results showed that sheath capillaries were located in the white pulp of the spleen, and the endothelial cells of sheath capillaries were cuboidal in shape, which is a typical characteristic of high endothelial venules. To monitor the lymphocyte homing, 5,6-carboxy fluoresceindiacetate succinimidyl ester (CFSE)-labeled lymphocytes that were intravenously injected into noninfected ducks appeared in the periellipsoidal sheaths (PELS), which proved that lymphocytes can return to the spleen through sheath capillaries. Furthermore, proteoglycans (PGs) associated with homing factors were positively observed in sheath capillaries and PELS by colloidal iron staining. This suggests that PGs are associated with lymphocyte homing. The results of the DTMUV infection experiment showed that PELS appeared vacuolized at 3 dpi. The spleen tissue gradually recovered at 5 and 7 dpi. In addition, the lymphocytes increased around sheath capillaries, and the expression of PGs in sheath capillaries increased after virus infection. Meanwhile, the gaps between endothelial cells were enlarged, and the lymphocytes were mainly in the lumen and basement membrane. In conclusion, lymphocytes could recruit into the spleen through sheath capillaries, and PGs participated and promoted the lymphocyte homing, suggesting that the unique high endothelial capillaries favor lymphocyte homing, which promotes tissue repair and antigen clearance in the duck.

Regulation of Macrophage and Dendritic Cell Function by Chondroitin Sulfate in Innate to Antigen-Specific Adaptive Immunity.

Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a linear acidic polysaccharide comprised of repeating disaccharides, modified with sulfate groups at various positions. Except for hyaluronan (HA), GAGs are covalently bound to core proteins, forming proteoglycans (PGs). With highly negative charges, GAGs interact with a variety of physiologically active molecules, including cytokines, chemokines, and growth factors, and control cell behavior during development and in the progression of diseases, including cancer, infections, and inflammation. Heparan sulfate (HS), another type of GAG, and HA are well reported as regulators for leukocyte migration at sites of inflammation. There have been many reports on the regulation of immune cell function by HS and HA; however, regulation of immune cells by CS has not yet been fully understood. This article focuses on the regulatory function of CS in antigen-presenting cells, including macrophages and dendritic cells, and refers to CSPGs, such as versican and biglycan, and the cell surface proteoglycan, syndecan.

ATDC5 cells as a model of cartilage extracellular matrix neosynthesis, maturation and assembly

Fibrillar collagens and proteoglycans (PGs) are quantitatively the major constituents of extracellular matrices (ECM). They carry numerous crucial post-translational modifications (PTMs) that tune the resulting biomechanical properties of the corresponding tissues. The mechanisms determining these PTMs remain largely unknown, notably because available established cell lines do not recapitulate much of the complexity of the machineries involved. ATDC5 cells are a model of chondrogenesis widely used for decades, but it remains described mostly at histological and transcriptional levels. Here, we asked to what extent this model recapitulates the events of ECM synthesis and processing occurring in cartilage. Insulin-stimulated ATDC5 cells exhibit up- or down-regulation of more than one-hundred proteins, including a number of known participants in chondrogenesis and major markers thereof. However, they also lack several ECM components considered of significant, yet more subtle, function in cartilage. Still, they assemble the large PG aggrecan and type II collagen, both carrying most of their in vivo PTMs, into an ECM. Remarkably, collagen crosslinking is fully lysyl oxidase (LOX)-dependent. The ATDC5 model recapitulates critical aspects of the cartilage ECM-processing machinery and should be useful to decipher the mechanisms involved.Proteomics data are available via ProteomeXchange with identifier PXD014121. SignificanceThe present work provides the first proteome characterization of the ATDC5 chondrogenesis model, which has been used for decades in the field of cartilage biology. The results demonstrate the up- and down-regulation of more than one hundred proteins. Overall, specific drawbacks of the model are pointed out, that will be important to take into consideration for future studies. However, major cartilage components are massively assembled into an extracellular matrix and carry most of their post-translational modifications occurring in cartilage tissue. Unlike other available established cell lines, the ATDC5 model recapitulates major aspects of cartilage biosynthesis and should be useful in investigating the mechanisms that regulate collagen maturation events.