Effect Of Proteoglycans Research Articles

Investigating the effect of proteoglycan 4 on hyaluronan solution properties using confocal fluorescence recovery after photobleaching

BackgroundThe objective of this study was to use confocal fluorescence recovery after photobleaching (FRAP) to examine the specific and dose-dependent effect of proteoglycan 4 (PRG4) on hyaluronan (HA) solutions of different molecular weight; and assess the effect of reduction and alkylation (R/A) of PRG4 on its effects on HA solutions.MethodsConfocal FRAP was used to determine the diffusion coefficient of fluorescein isothiocyanate (FITC)-dextran tracer (Dt) through 1500 kDa and 500 kDa HA solutions (0–3.3 mg/ml) ± PRG4 or a control protein, bovine serum albumin (BSA), at physiological (450 μg/ml) or pathophysiological (45 μg/ml) concentrations. The effect of PRG4 or R/A PRG4 on 1500 kDa HA solutions was also investigated. Empirical constants obtained from fitting data to the universal scaling equation were used to calculate the average distribution of apparent mesh sizes.ResultsPRG4 at both 45 and 450 μg/ml slowed the diffusion of the FITC-dextran tracer for all concentrations of HA and caused a decrease in the apparent mesh size within the HA solution. This effect was specific to PRG4, not observed with BSA, but not dependent on its tertiary/quaternary structure as the effect remained after R/A of PRG4.ConclusionsThese results demonstrate that PRG4 can significantly alter the solution properties of HA; PRG4 essentially reduced the permeability of the HA network. This effect may be due to PRG4 entangling HA molecules through binding and/or HA crowding PRG4 molecules into a self-assembled network. Collectively these findings contribute to the understanding of PRG4 and HA interaction(s) in solution and therefore the function of SF in diarthroidal joints.

Evaluation of the effect of salmon nasal proteoglycan on biomarkers for cartilage metabolism in individuals with knee joint discomfort: A randomized double-blind placebo-controlled clinical study.

A randomized double-blind placebo-controlled clinical trial was conducted to evaluate the chondroprotective action of salmon nasal cartilage proteoglycan on joint health. The effect of oral administration of proteoglycan (10 mg/day) on cartilage metabolism was evaluated in individuals with knee joint discomfort but without diagnosis of knee osteoarthritis. The average age of patients was 52.6±1.1 years old. The effect of proteoglycan was evaluated by analyzing markers for type II collagen degradation (C1,2C) and synthesis (PIICP), and the ratio of type II collagen degradation to synthesis. The results indicated that the change in C1,2C levels significantly differed in the proteoglycan group compared with the placebo group following 16 weeks intervention among subjects with high levels of knee pain and physical dysfunction (total score of Japan Knee Osteoarthritis Measure ≥41) and subjects with constant knee pain (both P<0.05). There was a greater increase in PIICP levels in the proteoglycan group than the placebo group following intervention, although this difference was not significant in both sets of patients. Thus, the C1,2C/PIICP ratios decreased in the proteoglycan group, whereas they slightly increased in the placebo group following the intervention. Furthermore, no test supplement-related adverse events were observed during the intervention. Therefore, oral administration of salmon nasal cartilage proteoglycan at a dose of 10 mg/day may exert a chondroprotective action in subjects with knee joint discomfort. This effect was achieved by improving cartilage metabolism (reducing type II collagen degradation and enhancing type II collagen synthesis), without causing apparent adverse effects.

Effect of proteoglycans at interfaces as related to location, architecture, and mechanical cues

IntroductionCovalently bound functional GAGs orchestrate tissue mechanics through time-dependent characteristics. ObjectiveThe role of specific glycosaminoglycans (GAGs) at the ligament–cementum and cementum–dentin interfaces within a human periodontal complex were examined. Matrix swelling and resistance to compression under health and modeled diseased states was investigated. Materials and methodsThe presence of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs at the ligament–cementum and cementum–dentin interfaces in human molars (N=5) was illustrated by using enzymes, atomic force microscopy (AFM), and AFM-based nanoindentation. The change in physical characteristics of modeled diseased states through sequential digestion of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs was investigated. One-way ANOVA tests with P<0.05 were performed to determine significant differences between groups. Additionally, the presence of mineral within the seemingly hygroscopic interfaces was investigated using transmission electron microscopy. ResultsImmunohistochemistry (N=3) indicated presence of biglycan and fibromodulin small leucine rich proteoglycans at the interfaces. Digestion of matrices with enzymes confirmed the presence of KS and CS GAGs at the interfaces by illustrating a change in tissue architecture and mechanics. A significant increase in height (nm), decrease in elastic modulus (GPa), and tissue deformation rate (nm/s) of the PDL-C attachment site (215±63–424±94nm; 1.5±0.7–0.4±0.2GPa; 21±7–48±22nm/s), and cementum–dentin interface (122±69–360±159nm; 2.9±1.3–0.7±0.3GPa; 18±4–30±6nm/s) was observed. ConclusionsThe sequential removal of GAGs indicated loss in intricate structural hierarchy of hygroscopic interfaces. From a mechanics perspective, GAGs provide tissue recovery/resilience. The results of this study provide insights into the role of GAGs toward conserved tooth movement in the socket in response to mechanical loads, and modulation of potentially deleterious strain at tissue interfaces.

In vitro friction testing of contact lenses and human ocular tissues: Effect of proteoglycan 4 (PRG4)

Contact lens friction was recently shown to correlate with in vivo comfort, with lower friction lenses providing improved comfort. Proteoglycan 4 (PRG4) is a recently discovered ocular surface boundary lubricant. The objectives of this study were to measure the friction of commercially available silicone hydrogel (SiHy) contact lenses against human cornea and eyelid tissues, and evaluate the ability of PRG4 to lubricate, and adhere to, SiHy contact lenses. The in vitro friction test employed here effectively measured and distinguished the SiHy contact lens friction coefficients against human eyelid and cornea tissues, and PRG4 functioned as an effective boundary lubricant.

Effect of dentin proteoglycans on the bonding properties of dentin

To investigate the potential effect of proteoglycan (PG) and glycosaminoglycan (GAG) on the bonding of etch and rinse adhesive to dentin, in order to improve the bonding effect of dentin. Forty-two extracted molars were used to obtain standard dentin bonding surface, and the specimens were etched for 15 s with 37% phosphoric acid and divided into three groups using a table of random numbers. Then the three groups undergone different incubating procedures as follow: specimens in chondroitinase ABC (C-ABC) group were incubated with C-ABC at 37 °C for 48 h in vibrator. Specimens in trypsin (TRY) group were incubated with trypsin, and specimens in the control group were incubated with deionized water for 48 h in the oscillators. Then specimens in each group were randomly assigned into two subgroups, A (Adper(TM) Single Bond 2) and B (Prime & Bond NT) (n = 7). The microtensile bond strength (µTBS), fracture mode and bonding interface morphology of the specimens were evaluated via microtensile testing, stereo microscope and field emission scanning electron microscopy (FE-SEM) respectively after specimens being incubated in 37 °C water for 24 h. The immediate µTBS of C-ABC group bonding with adhesive A and B [(32.9±2.5) and (26.8±2.2) MPa] were significantly lower than that of the control group [(40.7±3.3) and (34.6±3.7) MPa] (P < 0.05). While the immediate µTBS of TRY group [(49.0 ± 3.6) and (44.5 ± 3.0) MPa] were significantly higher than that of the control group(P < 0.05). Dentin PG participates in the dentin bonding process. Removal of PG increased the immediate µTBS of dentin and total etching adhesives, while removal of GAG decreased the immediate µTBS.

Effects of Proteoglycans on the Maturation of Dendritic Cells Derived from Human Peripheral Blood Monocytes

Dendritic cells (DCs) are a type of antigen-presenting cell which play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires maturation stimuli, such as pro-inflammatory cytokines or pathogen-derived components. Proteoglycans (PGs) are one of main components of the extracellular matrix and are composed of core proteins and glycosaminoglycans that bind to the core proteins. PGs are also constituent elements of bacteria and the role of PGs in the stimulation of DCs has not been elucidated. This study investigated the effects of PGs extracted from the nasal cartilage of a salmon head (S-PG) and the nasal septum cartilage of a whale (W-PG) on the maturation of DCs derived from human peripheral blood monocytes. This study was approved by the Committee of Medical Ethics of Hirosaki University School of Medicine. The human peripheral blood mononuclear cells (PBMCs) were separated from the buffy coat. Furthermore, the monocytes were separated from the PBMCs by allowing them to adhere to a plastic dish. To prepare iDCs, the monocytes were cultured in the presence of 50 ng/ml rhGM-CSF and rhIL-4 for 5 days. The iDCs were stimulated by S-PG or W-PG for 4 days to investigate whether the PGs alone were able to induce the maturation of DCs. In addition, other iDCs were stimulated by a cytokine mixture (rhTNF-α, rhIL-1β, rhIL- 6 and PGE2: MIX) or a combination of MIX+S-PG or W-PG for 48 hours. The surface phenotype of the DCs was analyzed by flow cytometry and the matrix metalloproteinase-9 (MMP-9) activity in the culture supernatants was assayed by zymography. Furthermore, the functions of DCs stimulated by a combination of MIX+S-PG or MIX+W-PG were examined. When the iDCs were stimulated by either S-PG or W-PG alone, the PGs-stimulated DCs did not express the DC-maturation marker CD83, thus indicating that S-PG and W-PG alone could not induce the maturation of DCs. However, the CCR5 expression on DCs stimulated by W-PG was down-regulated. When DCs were stimulated by MIX + 100 μg/ml W-PG, an up-regulation of CCR7 expression was observed. In association with the up-regulation of CCR7 expression, the stimulation by MIX+W-PG actually enhanced the chemotactic responsiveness of DCs to CCR7 ligand MIP-3β. These effects were not observed in the combination of MIX+S-PG. The MMP-9 activity was next examined by zymography, because the degradation of extracellular matrix by MMPs is required for DCs migration. However, neither S-PG nor W-PG promoted MMP-9 secretion. The present study therefore demonstrates that W-PG not but S-PG can selectively regulate the chemotactic activity of DCs in vitro. Further understanding of the mechanism and studies using human PGs is therefore expected to provide valuable insight into the migration of immune cells, including DCs both in vitro and in vivo.

Effects of Proteoglycan on Dextran Sulfate Sodium-Induced Experimental Colitis in Rats

Proteoglycans (PG) are macromolecules composed of glycosaminoglycan chains covalently attached to a protein core. In this study, we examined the effects of PG on dextran sulfate sodium (DSS)-induced experimental colitis in rats. First, to examine whether PG may ameliorate acute established DSS colitis, PG was administered orally for 5 days to the model animals. We evaluated the effects of PG on the basis of clinical symptoms, hematological analysis, macroscopic observation, and microscopic examination. We then examined whether PG administered orally to rats was detectable in their colonic lumen. After administration of PG, the colonic contents were collected, and the molecular weight of PG in the sample was analyzed by gel filtration high-performance liquid chromatography. Furthermore, we examined whether orally administered PG affected the concentrations of short-chain fatty acids (SCFAs) in the colonic feces. Orally administered PG ameliorated the clinical symptoms of bloody stools and diarrhea, and attenuated the increase in the white blood cell count in rats with established DSS colitis. Histologically, orally administered PG reduced the degree of mucosal erosion and inflammatory cell infiltration into the erosive area induced by DSS. Orally administered PG was detected in rat colon, although its molecular weight was slightly decreased. Orally administered PG significantly increased the concentration of total SCFAs and n-butyrate in rat colonic feces. This is the first study to indicate that exogenous PG ameliorates experimental colitis, suggesting the potential usefulness of PG for clinical treatment of colitis.

Effects of proteoglycans on the membrane dysfunction produced by in vitro ischemia in the rat hippocampal CA1 neurons.

Effects of proteoglycans on the membrane dysfunction produced by in vitro ischemia in the rat hippocampal CA1 neurons.

Adhesion of a chondrocytic cell line (USAC) to fibronectin and its regulation by proteoglycan.

Chondrocytes produce various extracellular matrices during chondrogenesis. Fibronectin and proteoglycan are major extracellular matrix proteins in cartilage tissue, but the interactions between them are not clear. Recently, we succeeded in establishing a cell line (USAC) with phenotypes of chondrocytes from a human osteogenic sarcoma of the mandible. Using this cell line, cell adhesion to fibronectin, the effect of proteoglycan on the cell adhesion and expression of integrin alpha5beta1 were investigated. Cells immediately adhered to fibronectin and then spread. Proteoglycan inhibited cell adhesion to fibronectin dose-dependently, whereas collagen did not. The expression of both mRNAs of alpha5 and beta1 subunits was detected 12 h after treatment with proteoglycan, but the expression of beta1 subunit mRNA had diminished by 24 h after treatment. These findings suggest that proteoglycan might modulate signal transduction from fibronectin by decreasing the expression of alpha5beta1 integrin.

Effect of proteoglycan on experimental colitis

The effect of proteoglycan (PG) on colitis was examined in animal experiments using mice. The PG used was extracted from nasal cartilage of salmon head with 4% acetic acid and prepared by precipitation with ethanol followed by dialysis. The PG contained about 7% protein, and had a molecular mass of 344 kDa on SDS/PAGE. The glycosaminoglycan (GAG) sugar chains of the PG were composed of hexosamine, uronic acid and sulfate at a molar ratio of 1.0:1.0:0.7. The mice were divided into a control group and an administration group. The control group was given free access to drinking water containing dextran sulfate sodium salt (DSS) to induce colitis. On the other hand, the administration group was given free access to drinking water containing DSS and PG. Then, the time course of survival rates in both groups were measured. In the administration group, the survival rate increased significantly in comparison with that of the control group. The difference in the survival rates indicated that the onset of mouse colitis induced by DSS was inhibited by administration of the PG.

Proteoglycans of the extracellular matrix and growth control.

Regulated cell growth results from the biological balance between soluble growth-regulating factors, their receptors and the elicited signal cascade on the one hand side and from extracellular macromolecular components and their interplay with membrane receptors on the other side. Proteoglycans have recently been recognized not only to play a part in providing shape and biomechanical strength of organs and tissues, but also to exhibit direct and indirect cell signalling properties. In this review, we discuss the direct growth-regulating role of proteoglycans with special emphasis on the lectican family and on the family of small proteoglycans with leucine-rich repeats (SLRPs). Indirect actions of proteoglycans by modulation of growth factor activities and growth factor distribution are exemplified by discussing the TGF-beta-binding properties of SLRPs and the interactions of core proteins of matrix proteoglycans with other growth factors. It is emphasized that the modulatory role of proteoglycans on cell proliferation cannot be separated from their participation in tissue organization in general, thereby explaining the diverse and sometimes contradictory reports on the effects of proteoglycans on cell proliferation and differentiation.

The effects of proteoglycan on GALT in rats treated with TPN.

It is well known that total parenteral nutrition (TPN) causes atrophy of the intestinal mucosa, resulting in degeneration and atrophy of the gut-associated lymphoid tissues (GALT). This study was conducted to examine the suppressive effect of TPN on GALT in rats. Rats that received TPN alone for 2 weeks, i.e., the TPN group, showed a decreased number of Peyer's patches and thoracic duct lymphocytes (TDL), as well as atrophy. Conversely, those treated with TPN in combination with polysaccharide K (PSK) at a daily dose of 1000 mg/kg for 2 weeks, i.e., the PSK group, showed increases in the number of Peyer's patches and TDL and improvement in the TDL subsets compared with the TPN group. Immunohistological examination of the changes in immunocytes in GALT using monoclonal antibodies revealed increases in the production of the major histocompatibility complex (MHC)-I and (MHC)-II, helper T cells, and interleukin 2 (IL-2). These findings indicate that PSK improves GALT suppression induced by TPN.

Trophic effect of collicular proteoglycan on neonatal rat retinal ganglion cells in situ

Naturally occuring neuronal death is widespread in the central nervous system of mammals. To date, the causes and mechanisms of such death are poorly understood. A major hypothesis is that developing neurons compete for limited amounts of trophic factor(s) released from their target centres as in the case of the peripheral nervous system and nerve growth factor. The present study aims to test this ‘trophic hypothesis’ in the mammalian central nervous system. In the rat, more than 50% of retinal ganglion cells die in the early post-natal period. Schulz and coworkers [57] purified a potential trophic agent from their major target, the superior colliculus, which was identified as a 480 kDa chondroitin sulfate proteoglycan. This proteoglycan or control solutions were injected into the eyes of rat pups during the post-natal part of the period of naturally occurring ganglion cell death. It was found that the collicular proteoglycan prevented the death of a significant number of the ganglion cells that would normally have been lost over a post-injection of one or two days. The effect of the proteoglycan was dose- and time-dependent. These results support the notion that trophic interactions are a determining factor in the survival of retinal ganglion cells during the period of naturally occurring cell death. It is also the first time that a proteoglycan has been shown to possess neurotrophic properties in situ.

Effect of proteoglycans on hydroxyapatite growth in vitro: the role of hyaluronan

The effect of cartilage proteoglycans on HA seed crystal growth was studied using a system providing constant supersaturation with respect to HA. The monomers were much less effective than the aggregates in reducing the rate of HA growth, which correlates with their affinity for the HA crystals. Hyaluronan, which is a normal constituent of the proteoglycan aggregates, behaved as a strong inhibitor of HA seed crystal growth and had an affinity constant similar to that of proteoglycan aggregates. The results indicate that inhibition of HA seed crystal growth is mediated throught the interaction of hyaluronan with HA crystal surface and that the proteoglycans add to the volume of the adsorbate causing steric hindrance.

Effect of proteoglycans on erythrocytes in the circulating blood

Effect of proteoglycans on erythrocytes in the circulating blood

Effects of Proteoglycan on Hydroxyapatite Formation Under Non-Steady-State and Pseudo-Steady-State Conditions

Addition of chondroitin sulfate (CS) or cartilage proteoglycan to metastable calcium phosphate solutions inhibits the formation of hydroxyapatite (HA). However, pre-equilibration of CS or proteoglycan with calcium prior to the addition of phosphate results in higher levels of HA precipitation compared to control solutions of identical calcium and phosphate activity. These findings indicate that the inhibition of HA formation by proteoglycans and CS is largely due to calcium binding. Further, its ability to bind calcium ions reversibly suggests that proteoglycan may act as a promoter, not an inhibitor, of calcification in cartilage.

The effects of selective matrix degradation on the short-term compressive properties of adult human articular cartilage

The effects of proteoglycan and collagen digestion on the transient response of human articular cartilage when tested in unconfined compression were determined. Small cylindrical specimens of cartilage, isolated from the femoral head of the hip joint and from the femoral condyles of the knee joint, were subjected to a suddenly applied compressive load using a test apparatus designed to yield a transient oscillatory response. From this response values of the elastic stiffness and the viscous damping coefficient were determined. Cathepsin D and cathepsin B 1 were used to digest the proteoglycan in some specimens, while in other specimens leukocyte elastase was used to attack the non-helical terminal regions of the Type II tropocollagen molecules and possibly the Type IX collagen molecule and thereby disturb the integrity of the collagen mesh. The results showed that proteoglycan digestion alone reduced the viscous damping coefficient but it did not significantly alter the elastic stiffness as determined from the oscillatory response. In contrast, the action of elastase reduced both the damping coefficient and the elastic stiffness of the cartilage. The results demonstrated the role of proteoglycans in regulating fluid transport in cartilage and hence controlling the time-dependent viscous properties. The elastic stiffness was shown to be dependent on the integrity of the collagen fibre network and not on the proteoglycans.

Effect of proteoglycans on lipoprotein-cell interactions: possible contribution to atherogenesis

Effect of proteoglycans on lipoprotein-cell interactions: possible contribution to atherogenesis

The Effect of Proteoglycans on the Morphology of Collagen Fibrils Formed In Vitro

The morphology of collagen fibrils at various times during formation in vitro was quantitatively examined by negative staining and by scanning electron microscopy. The presence of a small dermatan sulfate proteoglycan from bovine tendon (5 μg proteoglycan/100 μg collagen) resulted in collagen fibrils that were significantly thinner in width at all times by both methodologies. The rate of fibril diameter increase was also retarded by the small proteoglycans, suggesting that they inhibited the lateral aggregation of forming collagen fibrils. Large proteoglycans from cartilage did not produce this effect.

The effect of proteoglycans, collagen and lysyl oxidase on the metabolism of low density lipoprotein by macrophages

To study the interactions of lipoproteins, connective tissue components and cells, mouse peritoneal macrophages were incubated in the presence of human low density lipoproteins (LDL) that had been complexed with pig aortic proteoglycans (PG) or incubated in the presence of soluble collagen and/or lysyl oxidase, which catalyses the formation of cross-linkages in collagen and elastin by oxidising ϵ-amino groups of lysine residues to aldehydes. Soluble and insoluble PG-LDL complexes increased the incorporation of [ 3H]oleate into cellular cholesteryl esters (CE) 1.6- and 2.8-fold, respectively, while LDL incubated with collagen and lysyl oxidase had no effect compared to control LDL. As judged on the basis of incubations with fucoidin, spermine and 125I-labelled lipoproteins, the mechanism of internalisation of the PG-LDL complexes is different from that of acetylated LDL or dextran sulphate-LDL complexes. The formation of PG-LDL complexes in the arterial intima may lead to an increased uptake of lipoproteins by intimal macrophages during the early phase of atherogenesis.