Proteoglycan Research Articles

High fat-diet and saturated fatty acid palmitate inhibits IGF-1 function in chondrocytes.

Insulin-like growth factor-1 (IGF-1) promotes matrix synthesis and cell survival in cartilage. Chondrocytes from aged and osteoarthritic cartilage have a reduced response to IGF-1. The purpose of this study was to determine the effect of free fatty acids (FFA) present in a high-fat diet on IGF-1 function in cartilage and the role of endoplasmic reticulum (ER) stress. C57BL/6 male mice were maintained on either a high-fat (60% kcal from fat) or a low-fat (10% kcal from fat) diet for 4 months. Mice were then sacrificed; femoral head cartilage caps were collected and treated with IGF-1 to measure proteoglycan (PG) synthesis. Cultured human chondrocytes were treated with 500μM FFA palmitate or oleate, followed by stimulation with (100ng/ml) IGF-1 overnight to measure CHOP (a protein marker for ER stress) and PG synthesis. Human chondrocytes were pre-treated with palmitate or 1mM 4-phenyl butyric acid (PBA) or 1μM C-Jun N terminal Kinase (JNK) inhibitor, and IGF-1 function (PG synthesis and signaling) was measured. Cartilage explants from mice on the high fat-diet showed reduced IGF-1 mediated PG synthesis compared to a low-fat group. Treatment of human chondrocytes with palmitate induced expression of CHOP, activated JNK and inhibited IGF-1 function. PBA, a small molecule chemical chaperone that alleviates ER stress rescued IGF-1 function and a JNK inhibitor rescued IGF-1 signaling. Palmitate-induced ER stress inhibited IGF-1 function in chondrocytes/cartilage via activating the mitogen-activated protein (MAP) kinase JNK. This is the first study to demonstrate that ER stress is metabolic factor that regulates IGF-1 function in chondrocytes.

Effects of annulus defects and implantation of poly(lactic-co-glycolic acid) (PLGA)/fibrin gel scaffolds on nerves ingrowth in a rabbit model of annular injury disc degeneration

BackgroundGrowth of nerve fibers has been shown to occur in a rabbit model of intravertebral disc degeneration (IVD) induced by needle puncture. As nerve growth may underlie the process of chronic pain in humans affected by disc degeneration, we sought to investigate the factors underlying nerve ingrowth in a minimally invasive annulotomy rabbit model of IVD by comparing the effects of empty disc defects with those of defects filled with poly(lactic-co-glycolic acid)/fibrin gel (PLGA) plugs.MethodsNew Zealand white rabbits (n = 24) received annular injuries at three lumbar levels (L3/4, L4/5, and L5/6). The discs were randomly assigned to four groups: (a) annular defect (1.8-mm diameter; 4-mm depth) by mini-trephine, (b) annular defect implanted with a PLGA scaffold containing a fibrin gel, (c) annular puncture by a 16G needle (5-mm depth), and (d) uninjured L2/3 disc (control). Disc degeneration was evaluated by radiography, MRI, histology, real-time PCR, and analysis of proteoglycan (PG) content. Nerve ingrowth into the discs was assessed by immunostaining with the nerve marker protein gene product 9.5.ResultsInjured discs showed a progressive disc space narrowing with significant disc degeneration and proteoglycan loss, as confirmed by imaging results, molecular and compositional analysis, and histological examinations. In 16G punctured discs, nerve ingrowth was observed on the surface of scar tissue. In annular defects, nerve fibers were found to be distributed along small fissures within the fibrocartilaginous-like tissue that filled the AF. In discs filled with PLGA/ fibrin gel, more nerve fibers were observed growing deeper into the inner AF along the open annular track. In addition, innervations scores showed significantly higher than those of punctured discs and empty defects. A limited vascular proliferation was found in the injured sites and regenerated tissues.ConclusionsNerve ingrowth was significantly higher in PLGA/fibrin-filled discs than in empty defects. Possible explanations include (i) annular fissures along the defect and early loss of proteoglycan may facilitate the ingrowth process and (ii) biodegradable PLGA/fibrin gel may promote adverse growth of nerves and blood vessels into deeper parts of injured disc. The rabbit annular defect model of disc degeneration appears suitable to investigate the effects of nerve ingrowth in relation to pain generation.

Brain heparan sulphate proteoglycans are altered in developing foetus when exposed to in-utero hyperglycaemia.

In-utero exposure of foetus to hyperglycaemic condition affects the growth and development of the organism. The brain is one of the first organs that start to develop during embryonic period and glycosaminoglycans (GAGs) and proteoglycans (PGs) are one of the key molecules involved in its development. But studies on the effect of hyperglycaemic conditions on brain GAGs/PGs are few and far between. We, therefore, looked into the changes in brain GAGs and PGs at various developmental stages of pre- and post-natal rats from non-diabetic and diabetic mothers as well as in adult rats induced with diabetes using a diabetogenic agent, Streptozotocin. Increased expression of GAGs especially that of heparan sulphate class in various developmental stages were observed in the brain as a result of in-utero hyperglycaemic condition but not in that of adult rats. Changes in disaccharides of heparan sulphate (HS) were observed in various developmental stages. Furthermore, various HSPGs namely, syndecans-1 and -3 and glypican-1 were overexpressed in offspring from diabetic mother. However, in adult diabetic rats, only glypican-1 was overexpressed. The offsprings from diabetic mothers became hyperphagic at the end of 8weeks after birth which can have implications in the long run. Our results highlight the likely impact of the in-utero exposure of foetus to hyperglycaemic condition on brain GAGs/PGs compared to diabetic adult rats.

Membranes derived from human umbilical cord Wharton's jelly stem cells as novel bioengineered tissue-like constructs.

Cell-derived matrices were recently described as novel biomaterials generated by human cells allowed to grow and synthetize their own extracellular matrix in culture. In the present work, we generated and evaluated a novel tissue-like substitute (WDM) consisting of a membrane derived from cultured human Wharton's jelly stem cells. WDM were evaluated ex vivo and in vivo by histochemistry and immunohistochemistry for several mesenchymal cell markers and fibrillar and non-fibrillar extracellular matrix components. Results show that WDM were heterogeneous and consisted of dense cell-poor areas surrounded by cell-rich zones with abundant HWJSC. Histological analyses demonstrated that cell-poor areas were very rich in fibrillar and non-fibrillar extracellular matrix components such as collagen and proteoglycans, and cells in the WDM were highly viable and mostly PCNA-positive. HWJSC in the WDM expressed all markers of this cell type, including CD90, CD105, pan cytokeratin and CK8. In vivo analysis showed that the WDM was highly biocompatible and grafting this membrane in the muscle of laboratory rats was not associated to increased inflammation, necrosis, tumorigenesis or other side effects, while cells properly integrated at the damage site and showed high proliferation index. These results suggest that the structure and composition of the extracellular matrix of these novel WDM could reproduce the situation of native human tissues and that WDM implanted in vivo are highly biocompatible and rapidly integrate in the host tissues. For these reasons, we hypothesize that WDM could be used in regenerative medicine protocols.

Chondrodysplasia with multiple dislocations: comprehensive study of a series of 30 cases.

The group of chondrodysplasia with multiple dislocations includes several entities, characterized by short stature, dislocation of large joints, hand and/or vertebral anomalies. Other features, such as epiphyseal or metaphyseal changes, cleft palate, intellectual disability are also often part of the phenotype. In addition, several conditions with overlapping features are related to this group and broaden the spectrum. The majority of these disorders have been linked to pathogenic variants in genes encoding proteins implicated in the synthesis or sulfation of proteoglycans (PG). In a series of 30 patients with multiple dislocations, we have performed exome sequencing and subsequent targeted analysis of 15 genes, implicated in chondrodysplasia with multiple dislocations, and related conditions. We have identified causative pathogenic variants in 60% of patients (18/30); when a clinical diagnosis was suspected, this was molecularly confirmed in 53% of cases. Forty percent of patients remain without molecular etiology. Pathogenic variants in genes implicated in PG synthesis are of major importance in chondrodysplasia with multiple dislocations and related conditions. The combination of hand features, growth failure severity, radiological aspects of long bones and of vertebrae allowed discrimination among the different conditions. We propose key diagnostic clues to the clinician.

SRGN enhances the breast cancer cells metastasis by inducing Epithelial-mesenchymal transition

Objective To investigate the mechanism of glycoprotein serglycin (SRGN) promoting metastasis of breast cancer cells and the possible mechanism of SRGN expression. Methods Real time quantitative polymerase chain reaction (PCR) and bioinformation retrieval were used to detect the expression of SRGN in lymph node metastasis and non-metastasis breast cancer. MDA-MB-231shRNA and MCF-7-SRGN of breast cancer stable cell line were established by lentivirus shRNA interference and overexpression. Transwell assay was used to test the effect of SRGN on invasion and metastasis of breast cancer cell line in vitro. Western blot assay was used to detect the changes of epithelial-mesenchymal (EMT) related markers. The possible regulatory mechanism of SRGN expression was detected by Western blot assay. Results SRGN expression was significantly increased in lymph node metastasis of breast cancer in clinical specimens. SRGN interference inhibited the invasion and metastasis of tumor cells. SRGN promoted breast cancer cells EMT. Transforming growth factor β1 (TGFβ1) promoted the expression of beta SRGN transcription. Conclusions SRGN can induce the change of EMT in breast cancer cells and promote the invasion and metastasis of breast cancer cells. Key words: Proteoglycans; Breast neoplasms; Tumor cells, cultured; Biotransformation; Neoplasm invasiveness; Neoplasm metastasis

Scaffold-free approach produces neocartilage tissue of similar quality as the use of HyStem\u2122 and Hydromatrix\u2122 scaffolds

Numerous biomaterials are being considered for cartilage tissue engineering, while scaffold-free systems have also been introduced. Thus, it is important to know do the scaffolds improve the formation of manufactured neocartilages. This study compares scaffold-free cultures to two scaffold-containing ones. Six million bovine primary chondrocytes were embedded in HyStem™ or HydroMatrix™ scaffolds, or suspended in scaffold-free chondrocyte culture medium, and then loaded into agarose gel supported culture well pockets. Neocartilages were grown in the presence of hypertonic high glucose DMEM medium for up to 6 weeks. By the end of culture periods, the formed tissues were analyzed by histological staining for proteoglycans (PGs) and type II collagen, gene expression measurements of aggrecan, Sox9, procollagen α1(II), and procollagen α2(I) were performed using quantitative RT-PCR, and analyses of PG contents and structure were conducted by spectrophotometric and agarose gel electrophoretic methods. Histological stainings showed that the PGs and type II collagen were abundantly present in both the scaffold-free and the scaffold-containing tissues. The PG content gradually increased following the culture period. However, the mRNA expression levels of the cartilage-specific genes of aggrecan, procollagen α1(II) and Sox9 gradually decreased following culture period, while procollagen α2(I) levels increased. After 6-week-cultivations, the PG concentrations in neocartilage tissues manufactured with HyStem™ or HydroMatrix™ scaffolds, and in scaffold-free agarose gel-supported cell cultures, were similar to native cartilage. No obvious benefits could be seen on the extracellular matrix assembly in HyStem™ or HydroMatrix™ scaffolds cultures.Graphical abstract

Signal Transduction from the High Affinity IgE Receptor

Mast cells and basophils synthesize and store histamine, proteoglycans, and proteases in their granules and have surface receptors (FceRI) that bind IgE with high affinity. The interaction of multivalent antigen with this cell-bound IgE results in aggregation of FceRI and activation of the cells to release an array of inflammatory mediators. These mediators include the granular contents, arachidonic acid from phospholipids produced by the action of phospholipase enzymes, and cytokines such as tumor necrosis factor (TNF-α). Unlike the rapid release within minutes of preformed mediators, release of most cytokines is a slow process that requires several hours. This chapter reviews the current understanding of these intracellular pathways; most of this information is based on studies with the rat basophilic leukemia RBL-2H3 cultured mast cell line.

Glycosaminoglycan Sequence Influence on Iduronate Ring Puckering

Iduronate (IdoA) is a major monosaccharide constituent of the linear polysaccharides, called glycosaminoglycans (GAGs), that form covalent conjugates with core proteins to make proteoglycans (PGs). PGs are known as important structural components of extracellular matrix, and are increasingly recognized for their roles in cell signaling. Toward advancing understanding of the atomic-resolution structural biology of PGs, we have performed all-atom explicit-solvent molecular dynamics (MD) simulations on IdoA as a monosaccharide and in the context of oligosaccharides corresponding to fragments of the GAG dermatan sulfate (DS). The stereochemistry of the carbon atoms in the pyranose ring of IdoA and the resulting spatial distribution of functional groups attached to the ring result in a delicate thermodynamic balance between preferred ring puckering conformation for IdoA. Therefore, Cremer-Pople ring pucker analysis was applied to understand the sequence influence on IdoA in the context of DS oligosaccharide vs. IdoA monosaccharide. These results are compared with analogous simulations for glucuronate (GlcA), which is a single-site epimer (at C6) of IdoA, and makes up the related GAG chondroitin sulfate (CS).

Rigidity and flexibility in the tetrasaccharide linker of proteoglycans from atomic-resolution molecular simulation.

Proteoglycans (PGs) are covalent conjugates between protein and carbohydrate (glycosaminoglycans). Certain classes of glycosaminoglycans such as chondroitin sulfate/dermatan sulfate and heparan sulfate utilize a specific tetrasaccharide linker for attachment to the protein component: GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser. Toward understanding the conformational preferences of this linker, the present work used all-atom explicit-solvent molecular dynamics (MD) simulations combined with Adaptive Biasing Force (ABF) sampling to determine high-resolution, high-precision conformational free energy maps ΔG(φ, ψ) for each glycosidic linkage between constituent disaccharides, including the variant where GlcA is substituted with IdoA. These linkages are characterized by single, predominant (> 97% occupancy), and broad (45° × 60° for ΔG(φ, ψ) < 1 kcal/mol) free-energy minima, while the Xyl-Ser linkage has two such minima similar in free-energy, and additional flexibility from the Ser sidechain dihedral. Conformational analysis of microsecond-scale standard MD on the complete tetrasaccharide-O-Ser conjugate is consistent with ABF data, suggesting (φ, ψ) probabilities are independent of the linker context, and that the tetrasaccharide acts as a relatively rigid unit whereas significant conformational heterogeneity exists with respect to rotation about bonds connecting Xyl to Ser. © 2017 Wiley Periodicals, Inc.

Attenuation of obesity-induced inflammation in mice orally administered with salmon cartilage proteoglycan, a prophylactic agent

Obesity is associated with chronic inflammation of adipose tissue and causes development of type 2 diabetes. M1 macrophage population was increased in adipose tissue of obese mouse. M1 macrophages induce insulin resistance through the secretion of proinflammatory cytokines. Our previous studies demonstrated that salmon cartilage proteoglycan (PG) suppresses excess inflammation in various mouse inflammatory diseases. In this study, we examined the effect of PG on type 2 diabetes using high-fat-diet (HFD) induced obese mouse model. Oral PG administration enhanced the population of small adipocytes (area less than 1000 μm2) without body and tissue weight gain. In addition, PG administration suppressed mRNA expression of TNF-α, IL-6 and CXCL2 in adipose tissue. The proportion of M1 macrophages was decreased by PG administration. In addition, PG administration suppressed hyperglycemia after intraperitoneal glucose injection. Fasted serum insulin level was decreased in PG-administered mice. Moreover, insulin-stimulated phosphorylation of Akt was enhanced in the liver and gastrocnemius skeletal muscle of PG-administered mice. These data suggested that PG administration improves hyperglycemia and insulin sensitivity in obese mice by modulation of M1 macrophages which secrete proinflammatory cytokines in adipose tissue and activation of Akt in liver and skeletal muscle.

Contaminants in commercial preparations of 'purified' small leucine-rich proteoglycans may distort mechanistic studies.

The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial ‘decorin’ and ‘biglycan’ were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of ‘decorin’ and ‘biglycan’ on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers’ funds and time.

The Role of Endocan in the Development of Hepatocellular Carcinoma

Endocan is a dermatan sulfate proteoglycan, constituted of a core protein with 165 amino acids and a single dermatan sulfate chain covalently linked to the serine 137. It is mainly expressed by the activated endothelial cell and regulated by various cytokines and growth factors. In hepatocellular carcinoma (HCC), the expression of endocan increases significantly, and it can induce tumorigenesis and promote the tumor progression. Endocan involves in the proliferation of HCC cell, angiogenesis, metastasis and immunity via a variety of complicated pathways, which cover the regulation of hepatic inflammation-fibrosis-cancer (IFC) axis through nuclear factor-kappa B (NF-кB), modulating cell cycle, interaction with vascular endothelial growth factors (VEGF), the collapse of cell-cell junction, inhibiting of lymphocyte function associated antigen-1 /intercellular adhesion molecule-1(LFA-1/ICAM-1) and changing of tumor microenvironment. Therefore, it is a potential marker and promising therapy target of HCC.

Proteoglycans Isolated from Bramble Shark Cartilage (Echinorhinus brucus) Inhibits Proliferation of MCF-7 Human Breast Cancer Cells by Inducing Apoptosis

Proteoglycans (PGs) were isolated from deep sea shark Echinorhinus brucus (bramble shark) cartilage and their anti-proliferative activity against MCF-7 cell lines was evaluated. To establish apoptosis involvement for cytotoxicity, the following assays were performed i.e., caspases 3 and 9 assay, double fluorescent staining and DNA laddering assay. PGs were isolated by DEAE-sephacel ion exchange chromatography and identification and purity confirmed by chemical composition such as total carbohydrate, total protein, hexosamine, uronic aicd, sulfated GAG, wavelength scan and modified AGE. To confirm apoptosis pathway expression of genes BAX, Bcl-2 and p53 were carried out. PGs (100 μg/mL) induced a significant rise in activity of caspase 3 and 9, major apoptotic markers. A significant rise in the expression was also observed for Bcl-2 and p53. Whereas, low BAX expression was observed for PGs (100 μg/mL) treated sample. Significant anti-proliferative effect was observed and the mechanism behind cell death was concluded to be apoptosis.

Effect of temperature on photochemical and thermal changes in calf skin collagen solutions at physiological pH

Effect of temperature on photochemical and thermal changes in calf skin collagen solutions at physiological pH

Effects of pentosan polysulfate and polysulfated glycosaminoglycan on chondrogenesis of canine bone marrow-derived mesenchymal stem cells in alginate and micromass culture.

Mesenchymal stem cells (MSC) are a potential alternative source of differentiated chondrocytes for cartilage tissue regeneration and repair of osteoarthritic (OA) joints. We investigated the effects of pentosan polysulfate (PPS) and polysulfated glycosaminoglycan (PSGAG) on chondrogenesis of canine bone marrow-derived mesenchymal stem cells (cBMSC) in alginate and micromass cultures (MMC). Chondrogenic differentiation medium (CDM) was supplemented with PPS or PSGAG at concentrations of 0 (positive control; PC), 1, 3 and 5 µg/ml. 10% DMEM was used as negative control. Chondrocyte phenotype was analyzed by quantitative real-time PCR (qPCR) for alginate cultures and Alcian blue staining for proteoglycan (PG) synthesis for MMC. In alginate culture, PPS and PSGAG showed no significant effect on type II collagen, aggrecan and HIF-2α mRNA expression. PPS had no significant effect on type I collagen whereas PSGAG significantly upregulated (P<0.05) it at all concentrations relative to other treatments. PPS demonstrated a dose-dependent inhibitory effect on type X collagen mRNA with significant inhibition observed at 5 µg/ml compared to the NC. PSGAG showed an inverse effect on type X collagen with 1 µg/ml significantly inhibiting its expression while increase in the concentration correspondingly increased type X collagen expression. In MMC, PPS significantly enhanced chondrogenesis and PG deposition whereas PSGAG inhibited chondrogenesis and promoted a fibrocartilage-like phenotype with reduced PG deposition. While PPS enhances chondrogenesis of cBMSC in MMC, the response of MSC to chondroinductive factors is culture system-dependent and varies significantly between alginate and MMC.

Synthesis of a library of variously modified 4-methylumbelliferyl xylosides and a structure-activity study of human β4GalT7.

Proteoglycans (PGs) are complex macromolecules that are composed of glycosaminoglycan (GAG) chains covalently attached to a core protein through a tetrasaccharide linker. The biosynthesis of PGs is complex and involves a large number of glycosyltranferases. Here we present a structure-activity study of human β4GalT7, which transfers the first Gal residue onto a xyloside moiety of the linkage region. An efficient and regiocontrolled synthesis of a library of modified analogs of 4-methylumbelliferyl xyloside (XylMU) is reported herein. Hydroxyl groups at the position C-2, C-3 or C-4 have been epimerized and/or replaced by a hydrogen or a fluorine, while the anomeric oxygen was replaced by either a sulfur or a sulfone. The effect of these compounds on human β4GalT7 activity in vitro and on GAG biosynthesis in cellulo was then evaluated.

Structure-function relationships of human meniscus

Biomechanical properties of human meniscus have been shown to be site-specific. However, it is not known which meniscus constituents at different depths and locations contribute to biomechanical properties obtained from indentation testing. Therefore, we investigated the composition and structure of human meniscus in a site- and depth-dependent manner and their relationships with tissue site-specific biomechanical properties. Elastic and poroelastic properties were analyzed from experimental stress-relaxation and sinusoidal indentation measurements with fibril reinforced poroelastic finite element modeling. Proteoglycan (PG) and collagen contents, as well as the collagen orientation angle, were determined as a function of tissue depth using microscopic and spectroscopic methods, and they were compared with biomechanical properties. For all the measurement sites (anterior, middle and posterior) of lateral and medial menisci (n=26), PG content and collagen orientation angle increased as a function of tissue depth while the collagen content had an initial sharp increase followed by a decrease across tissue depth. The highest values (p<0.05) of elastic parameters (equilibrium and instantaneous moduli) and strain-dependent biomechanical parameters (strain-dependent fibril network modulus and permeability) were observed in the anterior horn of the medial meniscus. This location had also higher (p<0.05) PG content in the deep meniscus, higher (p<0.05) collagen content in the entire tissue depth, and lower (p<0.05) collagen orientation angle at the superficial tissue, as compared to many other locations. On the other hand, in certain comparisons (such as anterior vs. middle sites of the medial meniscus) significantly higher (p<0.05) collagen content and lower orientation angle, without any difference in the PG content, were consistent with increased meniscus modulus and/or nonlinear permeability. This study suggests that nonlinear biomechanical properties of meniscus, caused by the collagen network and fluid, may be strongly influenced by tissue osmotic swelling from the deep meniscus caused by the increased PG content, leading to increased collagen fibril tension. These nonlinear biomechanical properties are suggested to be further amplified by higher collagen content at all tissue depths and superficial collagen fibril orientation. However, these structure-function relationships are suggested to be highly site-specific.

Effect of selected flavonoids on glycosaminoglycans in human skin fibroblasts

Purpose: Glycosaminoglycans (GAGs) and proteoglycans (PG) in addition to collagen are the main components of extracellular matrix (ECM). They play an important role in intercellular communication and interactions between cells and ECM. The biological changes in ECM that occur during aging are induced by decrease in GAG biosynthesis. The purpose of this study was to evaluate the effect of selected flavonoids isolated from Cirsium palustre (L.) Scop. on GAG content in human skin fibroblasts. Materials and methods: Human skin fibroblasts were treated with eriodictyol 7-O-glucoside (C1), 6-hydroxyluteolin 7-O-glucoside (C2), scutellarein 7-O-glucoside (C3) and pedalitin (C4) at 1, 20 and 40 μM for 24 h. Concentration of GAGs in the medium was assayed using method based on their ability to bind the cationic dye 1,9- dimethylmethylene blue (DMMB). Results: C1, C2 and C4 at concentration of 20 and 40 µM significantly increased content of sulphated GAGs in the medium. In contrast, treatment of cells with compound C3 did not have a statistically significant impact on GAG level. Ascorbic acid used as a positive control at 50 µM showed no effect on GAG concentration and increased their content at 100 µM but to a much lower extent than flavonoids. Conclusion: Flavonoids C1, C2 and C4 showed greater than ascorbic acid stimulatory impact on GAGs in healthy human skin fibroblasts, demonstrating their therapeutic potential in the aging.

Chondroitin sulfate proteoglycans from salmon nasal cartilage inhibit angiogenesis

Because cartilage lacks nerves, blood vessels, and lymphatic vessels, it is thought to contain factors that inhibit the growth and development of those tissues. Chondroitin sulfate proteoglycans (CSPGs) are a major extracellular component in cartilage. CSPGs contribute to joint flexibility and regulate extracellular signaling via their attached glycosaminoglycan, chondroitin sulfate (CS). CS and CSPG inhibit axonal regeneration; however, their role in blood vessel formation is largely unknown. To clarify the function of CSPG in blood vessel formation, we tested salmon nasal cartilage proteoglycan (PG), a member of the aggrecan family of CSPG, for endothelial capillary-like tube formation. Treatment with salmon PG inhibited endothelial cell adhesion and in vitro tube formation. The anti-angiogenic activity was derived from CS in the salmon PG but not the core protein. Salmon PG also reduced matrix metalloproteinase expression and inhibited angiogenesis in the chick chorioallantoic membrane. All of these data support an anti-angiogenic role for CSPG in cartilage.