Proteoglycan Metabolism Research Articles

Proteoglycans in health and disease: emerging concepts and future directions

Proteoglycans are emerging as key molecules in a variety of cellular processes because of their unique capacities to regulate growth factor-mediated signaling pathways, thereby affecting many physiological and pathological processes, including, among others, cancer, angiogenesis, fibrosis, immunity and infectious diseases. The field has expanded so far and so fast that it is impossible to cover all proteoglycan biology. However, the four thematic minireviews provide the reader with a focused assessment of critical and emerging areas in ‘proteoglycanology’. The first minireview by Iozzo and Schaefer focuses on the role of the small leucine-rich proteoglycans (SLRPs) in cell signaling. Members of the SLRP gene family, now expanded to encompass five distinct classes, affect several receptor tyrosine kinases, including epidermal growth factor receptor, insulin-like growth factor receptor type 1 and hepatocyte growth factor receptor (Met). Often the interaction is antagonistic leading to receptor tyrosine kinase internalization and degradation, and ultimately to inhibition of cell growth. SLRPs have recently been linked to the regulation of factors in innate immunity and inflammation such as Toll-like receptors and purinergic P2X receptors. This is an expanding area of new research and suggests that SLRPs might have acquired a variety of functions through evolution. The second minireview by Manon-Jensen et al. focuses on the function of the cell-surface syndecans and their active enzymatic shedding. It is well established that proteolytic processing of cell-surface proteoglycans plays a key role in cell adhesion, migration, survival, differentiation and proliferation. Cleavage of the syndecan protein cores is constitutive, although this process is accelerated in wound healing and invasive cancer. Notably, shedding of the syndecans alters the cell-surface receptor dynamics and provides soluble ectodomains that can function as paracrine or autocrine effectors, or competitive inhibitors. The third minireview by Barash et al. focuses on new heparanase roles in cancer progression and metastasis. The activity of heparanase, an endo-β-d-glucuronidase which cleaves heparan sulfate chains at a limited number of sites, correlates with enhanced metastatic potential. Heparanase is also involved in angiogenesis, inflammation and autoimmunity, involving migration of vascular endothelial cells and activated cells of the immune system. Heparanase might also work in an enzymatic activity-independent manner, enhancing adhesion and migration of endothelial cells and promoting phosphorylation of signaling molecules such as Akt and Src. Human heparanase splice variants devoid of enzymatic activity and endowed with pro-tumorigenic characteristics may provide a mechanistic explanation for this function. The fourth minireview by Theocharis et al. focuses on the potential pharmacological targeting of proteoglycans in malignancy. The basic assumption is that proteoglycan metabolism is markedly altered during malignant transformation and progression, and that the synthesis of stromal proteoglycans is regulated by factors secreted by cancer cells. Thus, the modified tumor stroma might facilitate cancer cell growth and spreading, and the expression of proteoglycans in stromal cells may serve as prognostic markers for tumor progression and patient survival. We hope that this thematic minireview series will stimulate new studies in this interesting area of research and will attract new investigators to the field of proteoglycanology.

Proteoglycans in health and disease: novel roles for proteoglycans in malignancy and their pharmacological targeting

The expression of proteoglycans (PGs), essential macromolecules of the tumor microenvironment, is markedly altered during malignant transformation and tumor progression. Synthesis of stromal PGs is affected by factors secreted by cancer cells and the unique tumor-modified extracellular matrix may either facilitate or counteract the growth of solid tumors. The emerging theme is that this dual activity has intrinsic tissue specificity. Matrix-accumulated PGs, such as versican, perlecan and small leucine-rich PGs, affect cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Furthermore, expression of cell-surface-associated PGs, such as syndecans and glypicans, is also modulated in both tumor and stromal cells. Cell-surface-associated PGs bind various factors that are involved in cell signaling, thereby affecting cell proliferation, adhesion and motility. An important mechanism of action is offered by a proteolytic processing of cell-surface PGs known as ectodomain shedding of syndecans; this facilitates cancer and endothelial cell motility, protects matrix proteases and provides a chemotactic gradient of mitogens. However, syndecans on stromal cells may be important for stromal cell/cancer cell interplay and may promote stromal cell proliferation, migration and angiogenesis. Finally, abnormal PG expression in cancer and stromal cells may serve as a biomarker for tumor progression and patient survival. Enhanced understanding of the regulation of PG metabolism and the involvement of PGs in cancer may offer a novel approach to cancer therapy by targeting the tumor microenvironment. In this minireview, the implication of PGs in cancer development and progression, as well as their pharmacological targeting in malignancy, are presented and discussed.

Change in proteoglycan metabolism is a characteristic of human patellar tendinopathy

To determine differences in the metabolism of proteoglycans and the gene expression of proteinases and their inhibitors between patellar tendons exhibiting chronic overuse tendinopathy and normal patellar tendons in humans. Rates of loss and synthesis of proteoglycans were determined. Radiolabeled and total proteoglycans retained in and lost from the tissue were analyzed by fluorography and Western blotting. Levels of messenger RNA for matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-1, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, TIMP-3, and TIMP-4 were determined in fresh tissue. The rate of loss of (35)S-labeled proteoglycans was greater in abnormal tendons, as was the rate of synthesis of proteoglycans. Fluorography and Western blotting revealed the presence of greater amounts of large proteoglycans (aggrecan and versican) in abnormal tendons, and these proteoglycans were rapidly lost from the matrix of abnormal tendons. There was no significant difference in the expression of ADAMTS-1, ADAMTS-4, ADAMTS-5, MMP-1, MMP-2, MMP-3, MMP-13, TIMP-2, TIMP-3, or TIMP-4. There was a significant increase in the expression of MMP-9 and TIMP-1 in abnormal tendons. Our findings suggest that a change in the proteoglycan content of the extracellular matrix in abnormal tendons results from the altered metabolism of the cells, reflected in the enhanced synthesis of the large proteoglycans aggrecan and versican, and does not appear to result from changes at the level of gene expression.

The proteoglycan metabolism of articular cartilage in joint-scale culture.

Understanding and controlling chondrocyte and cartilage metabolism in osteochondral tissues may facilitate ex vivo maintenance and application, both for allografts and tissue-engineered grafts. The hypothesis of this study was that maintenance of chondrocyte viability and matrix content and release of sulfated glycosaminoglycan (sGAG) in the articular cartilage of joint-scale osteochondral fragments are temperature and metabolism dependent. The aims were to assess, for adult goat joints, the effects of incubation temperature (37 degrees C vs. 4 degrees C) on cartilage chondrocyte viability and tissue matrix content and mechanical function, and the effects of temperature and cellular biosynthesis on sGAG release. Chondrocyte viability was maintained with 37 degrees C incubation for 28 days, but decreased by approximately 30% with 4 degrees C incubation. Concomitantly, with 37 degrees C incubation, cartilage sGAG was depleted by approximately 52% with the lost sGAG predominantly unable to aggregate with hyaluronan, whereas collagen content, tissue thickness, and tissue stiffness were maintained. The depletion of sGAG was diminished by slowing metabolism, with 4 degrees C decreasing release by approximately 79% compared with 37 degrees C incubation, and cycloheximide inhibition of cell metabolism at 37 degrees C decreasing release by approximately 47%. These results indicate that the articular cartilage of joint-scale grafts have enhanced chondrocyte viability with incubation at 37 degrees C, but may need anabolic stimuli or catabolic inhibitors to maintain sGAG content.

Insulin-dependent Diabetes Mellitus in Mice Does Not Alter Liver Heparan Sulfate

Diabetes -associated hyperlipidemia is generally attributed to reduced clearance of plasma lipoproteins, especially remnant lipoproteins enriched in cholesterol and triglycerides. Hepatic clearance of remnants occurs via low density lipoprotein receptors and the heparan sulfate proteoglycan, syndecan-1. Previous studies have suggested alterations in heparan sulfate proteoglycan metabolism in rat and mouse diabetic models, consistent with the idea that diabetic dyslipidemia might be caused by alterations in proteoglycan expression in the liver. In this study we analyzed the content and composition of liver heparan sulfate in streptozotocin-induced insulin-deficient diabetic mice that displayed fasting hypertriglyceridemia and delayed clearance of dietary triglyceride-rich lipoproteins. No differences between normal and diabetic littermates in liver heparan sulfate content, sulfation, syndecan-1 protein levels, or affinity for heparin-binding ligands, such as apolipoprotein E or fibroblast growth factor-2, were noted. Decreased incorporation of [(35)S]sulfate in insulin-deficient mice in vivo was observed, but the decrease was due to increased plasma inorganic sulfate, which reduced the efficiency of labeling of liver heparan sulfate. These results show that hyperlipidemia in insulin-deficient mice is not due to changes in hepatic heparan sulfate composition.

Involvement of a cysteine protease in the secretion process of human xylosyltransferase I

Xylosylation of core proteins takes place in the Golgi-apparatus as the transfer of xylose from UDP-xylose to specific serine residues in proteoglycan core proteins. This initial and rate-limiting step in glycosaminoglycan biosynthesis is catalyzed by human xylosyltransferase I (XT-I). XT-I is proteolytically cleaved from the Golgi surface and shed in its active form into the extracellular space. The secreted, circulating glycosyltransferase represents a serum biomarker for various diseases with an altered proteoglycan metabolism, whereas a physiological function of secreted XT-I is still unknown. To shed light on the secretion process of XT-I and on its biological function, the cleavage site was examined and the group of proteases involved in the cleavage was identified in this study. The peptide mass fingerprint from partly purified secreted XT-I revealed the cleavage site to be localized in the aminoterminal 231 amino acids. The addition of a cysteine protease inhibitor cocktail to cells recombinantly expressing XT-I led to a concentration-dependent shift of enzyme activity towards the cell lysates attended by consistent total intracellular and extracellular XT-I activities. In conclusion, our findings provide a first insight into the XT-I secretion process regulated by a cysteine protease and may contribute to understanding the biological and pathological role of this process.

Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitroeffects on cartilage metabolism and intra-articular levels after injection

IntroductionIntraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.MethodsThe effect of conditioned serum on PG metabolism of cultured OA cartilage explants was compared to unconditioned serum. The effect of serum conditioning on levels of interleukin-1beta (IL-1β), IL-4, IL-6, IL-10, IL-13, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), osteoprotegerin (OPG), oncostatin M (OSM), interleukin-1 receptor (IL-1ra) and transforming growth factor beta (TGF-β) were measured by multiplex ELISA. As TNF-α levels were found to be increased in conditioned serum, the effect of TNF-α inhibition by etanercept on PG metabolism was studied in cartilage explants cultured in the presence of conditioned serum. Furthermore, cytokine levels in SF were measured three days after intraarticular ACS injection in OA patients to verify their retention time in the joint space.ResultsPG metabolism was not different in the presence of conditioned serum compared to unconditioned serum. Levels of the anti-inflammatory cytokines IL-1ra, TGF-β, IL-10 as well as of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and OSM were increased. IL-4, IL-13 and IFN-γ levels remained similar, while OPG levels decreased. TNF-α inhibition did not influence PG metabolism in cartilage explant culture in the presence of condtioned serum. Although OPG levels were higher and TGF-β levels were clearly lower in ACS than in SF, intraarticular ACS injection in OA patients did not result in significant changes in these cytokine levels.ConclusionsACS for treatment of osteoarthritis contains increased levels of anti-inflammatory as well as pro-inflammatory cytokines, in particular TNF-α, but conditioned serum does not seem to have a net direct effect on cartilage metabolism, even upon inhibition of TNF-α. The fast intraarticular clearance of cytokines in the injected ACS may explain the limited effects found previously in vivo.

Chondroprotective effects of low-frequency lowenergy pulsed electromagnetic fields on human cartilage explants

Objective To investigate the effects of low-frequency low-energy pulsed electromagnetic fields (PEMFs) on proteoglycans (PG) metabolism [1-2] of human articular cartilage explants derived from patients with different osteoarthritis (OA) grades in the presence and in the absence of anabolic and catabolic stimuli for cartilage. Design Human knee cartilage explants derived from lateral and medial femoral condyles of OA patients were exposed to PEMF (1.5 mT; 75 Hz) for 1 and 7 days with and without insulin-like growth factor-I (IGF-I) (50 ng/ml) and interleukin-1β (IL-1β) (0.01-50ng/ml). Histological analysis was used to classify cartilage according to the score developed by the International Cartilage Repair Society (ICRS). PG synthesis was determined by 35S-sulfate incorporation. PG release into culture media was determined by the dimethylmethylene blue assay. Results By histological evaluation, cartilage explants derived from lateral and medial condyles were classified as OA ICRS grade I and III, respectively. After 7 days treatment, in OA grade I cartilage explants, PEMF and IGF-I alone significantly increased 35S-sulfate incorporation respect to control and counteracted the effect induced by IL-1β (0.01 ng/ml) of 26% and 24% respectively. The combination of PEMF and IGF-I resulted additive in all conditions. Similar results were obtained in OA grade III cartilage, although basal 35S-sulfate incorporation was lower than in OA grade I. No effect was observed on medium PG release. Conclusions PEMF exerts chondroprotective effects on human articular cartilage. The biological responsiveness of chondrocyte to PEMF was similar in different OA grades cartilage, suggesting that human chondrocytes maintain susceptibility to PEMF during OA progression.

A Phenotypic Comparison of Proteoglycan Production of Intervertebral Disc Cells Isolated from Rats, Rabbits, and Bovine Tails; Which Animal Model is Most Suitable to Study Tissue Engineering and Biological Repair of Human Disc Disorders?

The nucleus pulposus (NP) of the intervertebral disc in cattle and humans shows the most dramatic changes with aging of any cartilaginous tissue. In humans, notochordal cells disappear from the NP and are replaced with chondrocytic cells by adolescence. However, notochordal cells of the NP persist into adult life in some species, such as rats and rabbits. Therefore, comparison of the metabolic activity of notochordal and nonnotochordal cells is considered to be important for determining the type of cell to use for transplantation to regenerate intervertebral discs. In this study, we investigated the notochordal NP cells of rats and rabbits, as well as nonnotochordal (chondrocyte-like) bovine NP cells, in a three-dimensional culture system to examine whether proteoglycan metabolism varied among these three cell types. As a result, bovine NP cells produced around 0.18 mg/mL of glycosaminoglycan after culture for 5 days, while rat and rabbit NP cells produced about four and two times more glycosaminoglycan than bovine cells, respectively. In conclusion, this study demonstrated marked differences of energy metabolism and production of matrix components between notochordal and nonnotochordal NP cells. Animals with notochordal cells in the NP, such as rats and rabbits, may not provide good models for investigation of biological repair and tissue engineering for human disc disorders.

Lidocaine Cytotoxicity to the Zygapophysial Joints in Rabbits

STUDY DESIGN.: To examine whether lidocaine cytotoxicity to chondrocytes has been implicated in the development of osteoarthritis of the zygapophysial joints. OBJECTIVE.: This study was performed to determine the effects of varying concentrations and exposure times of lidocaine on the viability and proteoglycan metabolism of rabbit zygapophysial chondrocytes in vitro. SUMMARY OF BACKGROUND DATA.: Zygapophysial joint injections are commonly administered with lidocaine for chronic spinal pain in orthopedic treatment. A lot of studies on the effect of zygapophysial joint injections are clinical, but many questions on the effect of lidocaine to zygapophysial chondrocytes remain unanswered. METHODS.: Cartilage was obtained from zygapophysial joints of adult rabbits. Chondrocytes in alginate beads were cultured in medium containing 6% fetal calf serum at 370 mOsmol at cell densities of 4 million cells/mL. They were then cultured for 24 hours under 21% oxygen with 0.125%, 0.25%, 0.5%, and 1% lidocaine, and without lidocaine as control. The cell viability profile across intact beads was determined by manual counting using fluorescent probes (LIVE/DEAD assay) and transmission electron microscopy. Lactate production was measured enzymatically as a marker of energy metabolism. Glycosaminoglycan (GAG) accumulation was measured using a modified dimethylmethylene blue assay. RESULTS.: Cell viability decreased in a time- and dose-dependent manner in the concentration range of 0.125% to 1.0% lidocaine under the confocal microscope. Under the electron microscope, apoptosis increased as the concentration of lidocaine increased. GAG accumulation/tissue volume decreases as the concentration of lidocaine increased. However, GAG produced per million cells and the rate of lactate production per live cell was significantly higher for cells cultured at 0.5% and 1% lidocaine than the control group. CONCLUSION.: While these in vitro results cannot be directly extrapolated to the clinical setting, this data suggestcaution in prolonged exposure of zygapophysial cartilage to high concentration lidocaine.

Metabolism of proteoglycans in tendon

Tendons are dense, fibrous connective tissues that are responsible for transmitting mechanical forces from skeletal muscle to bone. From a clinical perspective, tendinopathy (defined as a syndrome of tendon pain, tenderness and swelling that affects function) is very common, both within the sporting arena and in the workplace. Importantly, proteoglycans are essential components of the tendon extracellular matrix (ECM) and changes in their expression and metabolism/turnover have been associated with tendinopathy. Within tendons, the small leucine-rich proteoglycans (SLPRs), decorin, fibromodulin, lumican and keratocan predominate within tensional regions, while in tendon fibrocartilage, increased concentrations of proteoglycans common to the articular cartilage phenotype are present, including aggrecan, biglycan and proteoglycan 4. However, the rate of proteoglycan turnover within tendon is markedly higher than that of cartilage, mediated via the "aggrecanases," which are constitutively active in the tendon matrix. Data suggest that this increased proteoglycan turnover is likely to be required to maintain normal tendon homeostasis, with perturbations in proteoglycan metabolism contributing to tissue dysfunction. Thus, future studies aimed at furthering our fundamental knowledge of tendon proteoglycan metabolism in health and disease are important in the development of improved treatments for tendon disorders.

Identification of a xylosyltransferase II gene haplotype marker for diabetic nephropathy in type 1 diabetes

Background Proteoglycans are major components of the glomerular basement membrane, being responsible for their permeability properties. Type 1 diabetic patients have an altered proteoglycan metabolism, which contributes to microvascular complications like diabetic nephropathy. Xylosyltransferase II (XT-II) is a chain-initiating enzyme in the biosynthesis of basement membrane proteoglycans and catalyzes the transfer of xylose to selected serine residues in the core protein. Thus, genetic variations in the XT-II coding gene XYLT2 might be implicated in the initiation and progression of late diabetic complications. Methods Genotyping of 6 genetic variations in the XYLT2 gene and haplotype analysis was performed in 697 type 1 diabetic patients (358 with and 338 without diabetic nephropathy). Results The haplotype analysis of 6 XYLT2 polymorphisms revealed one haplotype (GATTCG) to be significantly less frequent among type 1 patients with diabetic nephropathy ( p = 0.002, OR = 0.13, 95% CI = 0.03–0.59). The haplotype GATTCG consist of the XYLT2 variations c.166G>A, c.177A>G, c.342T>C, IVS6-9T>C, c.1569C>T and c.2402C>G. No genotype–phenotype interactions were revealed. Conclusions Our data show that a XYLT2 haplotype is associated with nephropathy in type 1 diabetic patients.

Serum Xylosyltransferase Activity in Diabetic Patients as a Possible Marker of Reduced Proteoglycan Biosynthesis

OBJECTIVE—Proteoglycan metabolism is altered in diabetic patients. The xylosyltransferases (XTs) are the initial and rate-limiting enzymes in the biosynthesis of the glycosaminoglycan chains in proteoglycans. Here, we analyzed whether the changed proteoglycan metabolism leads to altered serum XT levels in diabetic patients.RESEARCH DESIGN AND METHODS—Serum XT activity was determined in 100 diabetic patients and 100 blood donors using a novel high-performance liquid chromatography electrospray ionization tandem mass spectrometry assay.RESULTS—Serum XT activities in male and female diabetic patients were significantly decreased compared with those in the corresponding normoglycemic control subjects (mean ± SD: male patients, 19.3 ± 4.44 mU/l; male nondiabetic control subjects, 26.6 ± 2.79 mU/l; female patients, 18.9 ± 3.14 mU/l; female nondiabetic control subjects, 21.8 ± 3.74 mU/l; P < 0.0001). No significant differences were detected between patients with type 1 and type 2 diabetes.CONCLUSIONS—Our data show decreased XT activity in patients with diabetes, a disease that is accompanied by an altered proteoglycan biosynthesis.

Morphological and biochemical studies of the cartilage in the diabetic rat--a model for metabolism-induced arthritis

Because of its general effects on connective tissue metabolism diabetes mellitus is becoming more and more important in the orthopaedic field. For investigation of morphologic changes in the cartilage a model of streptozotocin-induced diabetes was used in 80 rats. Studies were performed by light-, scanning- and transmission-electron microscopy and by biochemical analysis of proteoglycan and collagen metabolism. Three months after onset of diabetes the chondrocytes show increased metabolic activity and at the same time first stage regressive changes. The morphologic changes correlate with a significant decrease of hexosamine content in the cartilage. There is no evidence for any disturbance of collagen metabolism at that time. After seven months the cartilage is widely necrotic, the collagen fibers are swollen and show almost total loss of their microstructure. There is no sign for reparation of the necrotic zones at this time. It is most remarkable that in many specimen crystal deposits are found just below the cartilage surface which from the TEM-findings look very much like calciumpyrophosphate crystals. The morphologic and biochemical results show that in the rat diabetes leads to a special type of cartilage changes with crystallic arthropathy which seems to be a valuable model for the study of metabolic osteoarthrosis.

Biological Activities of Exogenous Polysaccharides &lt;i&gt;via&lt;/i&gt; Controlling Endogenous Proteoglycan Metabolism in Vascular Endothelial Cells

Proteoglycan contains glycosmainoglycans, which are endogenous sulfated polysaccharides, in the molecule. The metabolism of proteoglycans regulates cell behavior and cellular events. It is possible that exogenous polysaccharide-related molecules exhibit their biological activities by two mechanisms. One is the interaction with cells and the other is the interaction with growth factors/cytokines that regulate proteoglycans. In this review, we describe sodium spirulan, a sulfated polysaccharide obtained from a hot-water extract of the blue-green alga Spirulina platensis, as an exogenous polysaccharide that stimulates the release of proteoglycans from vascular endothelial cells. Factors that regulate endothelial proteoglycan metabolism are also being described as possible target molecules of exogenous polysaccharides. Further research is required to obtain exogenous polysaccharide-related molecules that exhibit useful biological activities through controlling endothelial proteoglycan metabolism for protection against vascular lesions such as atheroslcerosis.

Development of Partial-thickness Articular Cartilage Injury in a Rabbit Model

In humans, partial-thickness cartilage lesions frequently result in premature osteoarthritis. While rabbits often are used as a model for partial-thickness cartilage lesions, the natural course of cartilage surrounding such a lesion is largely unknown. We developed a rabbit model of a chronic partial-thickness cartilage defect and asked whether these defects led to (1) deterioration of surrounding cartilage macroscopically and microscopically (increased Mankin score) and (2) disturbances in proteoglycan metabolism. In 55 rabbits, we created a 4-mm-diameter partial-thickness cartilage defect on one medial femoral condyle. The surrounding cartilage was characterized during the course of 26 weeks. Contralateral knees were sham-operated. In experimental knees, we found cartilage softening and fibrillation at 13 and 26 weeks. High Mankin scores observed at 1 week were partially restored at 13 weeks but worsened later and were most pronounced at 26 weeks. Mankin scores in the experimental groups were worse at 1 and 26 weeks when compared with the sham groups. Mankin scores at 26 weeks improved compared with 1 week in the sham groups. Disturbances in proteoglycan metabolism were less evident. In this rabbit model, a partial-thickness cartilage lesion resulted in early markers of degenerative changes resembling the human situation.

Transient Increase with Strenuous Exercise of Plasma Levels of Glycosaminoglycans in Humans and Horses

Plasma glycosaminoglycans (GAGs) were isolated and purified by chromatographic procedures in healthy humans and horses before and after physical exercise. A weak anion exchange resin was used to separate polyanions. Humans exercised on a cycloergometer, while horses were exercised on a treadmill and in show jumping competition. Some GAGs were isolated from untreated plasma and operationally defined as native proteoglycans (PGs), while the total GAG amount was isolated from mild alkali treated plasma. Plasma GAG concentrations in terms of galactosamine and galactose, the respective components of the cartilage polysaccharides chondroitin sulphate and keratan sulphate, significantly increased only when the subject was exercised intensively, in both humans and horses. Only native PGs fraction contributed to GAG levels changes. The increases were transient, since preexercise values were reached in 30 min in horses. These changes with exercise are suggested to be a tool to measure the effect of physical exercise on cartilage PG metabolism.

Effects of Reloading After Simulated Microgravity on Proteoglycan Metabolism in the Nucleus Pulposus and Anulus Fibrosus of the Lumbar Intervertebral Disc

An experiment to measure proteoglycan (PG) content and PG-related gene mRNA expressions in the lumbar intervertebral disc (IVD) of rats tail-suspended (TS) for up to 6 weeks with subsequent reloading. To assess the effects of reloading after simulated microgravity on PG metabolism in nucleus pulposus (NP) and anulus fibrosus (AF). Although the PG content of rat lumbar IVD is reportedly decreased by low compressive force (due to so-called microgravity) during spaceflight, it is unknown whether it recovers completely on reloading and whether these effects differ between NP and AF. Eighty-five F344/N rats were divided as follows: caged control (C) or TS for either 3 or 6 weeks, with some TS rats reloaded for 1 or 2 days or 3 weeks after 3 weeks' suspension (TS+RL-1d, -2d, or -3w). The glycosaminoglycan content and mRNA levels for aggrecan, TIMP1, MMP3, and ADAMTS4 were measured in NP and AF. The glycosaminoglycan contents of NP and AF were significantly decreased (by 27%-42%) in the TS groups, whereas in the TS+RL-3w group recovery was complete in NP, but incomplete in AF, without histologic degenerative changes at any time point. In NP, the aggrecan mRNA level was significantly downregulated in TS-3w, but recovered to control level on reloading (TS+RL-3w). In AF, the MMP3 mRNA level was significantly elevated in TS-6w. In the early (1-2 days) response of PG-related gene expressions to reloading, mRNA levels were significantly increased for aggrecan, TIMP1, and ADAMTS4 in NP and for MMP3 in AF, but significantly decreased for ADAMTS4 in AF (vs. the TS-3w group). Our results suggest that in IVD maintenance against the present type of mechanical stress, modulation of PG plays an important role and may be associated with molecular changes in PG-related genes.

The quantitative and functional relation between insulin‐like growth factor‐I (IGF) and IGF‐binding proteins during human osteoarthritis

A previous hypothesis stated that during osteoarthritis (OA) increased insulin-like growth factor (IGF) binding proteins (IGFBPs) sequester IGFs and limit their access to the cell. The objective of this article was to test this by: (1) quantifying IGF and IGFBP-3 as well as their ratios in human OA cartilages, and (2) measuring the metabolic responses of diseased cartilage to IGF-I and its IGFBP-insensitive analogs. Knee or hip OA cartilages were staged for OA by histology. Cartilage slices were either extracted for assays of IGF proteins, or maintained intact as organ cultures. Proteoglycan (PG) metabolism +/- IGFs was measured by use of the (35)S-sulfate precursor. IGFBP-3 (ng/mg protein) was weakly correlated with OA score by regression analysis (R(2) = 0.122; p = 0.040; n = 35). IGF-I (ng/mg protein) was constant across all OA groups (ANOVA; p = .428, n = 18) and the IGF-I/IGFBP-3 ratios were > 1 in most samples. All OA cartilages responded to hrIGF-I by increasing PG synthesis [average 2.29-fold +/- 0.55 (+/-SD) at saturation, n = 12] irrespective of OA score. The des (1-3) IGF-I analog (which lacks the three N-terminal amino acids) had similar maximal effects (average 2.23-fold stimulation +/- 0.71, n = 10), but it was more effective in two out of three samples at suboptimal doses. The effect of hrIGF-I, des (1-3) IGF-I, or the B-chain analog on degradation was minimal. In summary, catabolism was insensitive to IGF-I, and this was probably not due to IGFBPs. By contrast, IGF-I exerted a robust stimulation of anabolism at sufficiently high doses, even though IGFBPs could tone down the ligand effect at low doses.

Articular cartilage metabolism in patients with Kashin–Beck Disease: an endemic osteoarthropathy in China

The objective of this study was to investigate CD44 and proteoglycan metabolism in patients suffering from Kashin-Beck Disease (KBD), an endemic osteoarthropathy that affects 2.5 million of 30 million people living in the KBD regions of China. Immunohistochemical analyses of cluster of differentiation-44 (CD44), BC-13 and 3-B-3(-) expression were performed in cartilage sections harvested from KBD and normal patients. In addition, the serum levels of soluble CD44 (sCD44), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase-1 were determined using a sandwich enzyme linked immunosorbent assay. Hematoxylin & eosin and toluidine blue staining indicated that there was cell necrosis and proteoglycan loss in cartilage from both KBD children and adult cartilage. Strong immunohistochemical staining for CD44, BC-13 and 3-B-3(-) occurred in the majority of adult KBD patients and most KBD children. Furthermore, statistically significant elevated levels of sCD44, IL-1beta and TNF-alpha were found in the sera of both adult and child KBD patients when compared to the levels of normal adult and child controls. Interestingly, IL-1beta and TNF-alpha serum levels were all high in normal children from KBD regions when compared to normal children from non-KBD regions suggesting that unidentified factors (e.g., a genetic predisposition) may protect some people from KBD pathology. Our results demonstrate that altered CD44, IL-1beta and TNF-alpha metabolism occurs in the pathogenesis of KBD and there is an increased aggrecanase-generated proteoglycan loss from KBD adult and child cartilage. These primary metabolic changes are likely to be significant contributing factor causing pathological joint formation and instability that leads to secondary osteoarthritis in KBD patients.