The effects of oral glucosamine on joint health: is a change in research approach needed?

OBJECTIVE: Oral glucosamine (GlcN) has been widely studied for its potential therapeutic benefits in alleviating the pain and disability of osteoarthritis (OA). Its popularity has grown despite ongoing controversy regarding its effectiveness vs placebo in clinical trials, and lack of information regarding possible mechanisms of action. Here, we review the state of knowledge concerning the biology of GlcN as it relates to OA, and discuss a framework for future research directions. METHODS: An editorial "narrative" review of peer-reviewed publications is organized into four topics (1) Chemistry and pharmacokinetics of GlcN salts (2) Biological effects of GlcN salts in vitro (3) Therapeutic effects of GlcN salts in animal models of OA and (4) GlcN salts in the treatment of clinical OA. RESULTS: Data reporting potent pleiotropic activities of GlcN in in vitro cell and explant cultures are discussed in the context of the established pharmacokinetic data in humans and animals. The available clinical trial data are discussed to place the patient in the context of controlled research on disease management. CONCLUSIONS: Future research to determine therapeutic mechanisms of GlcN salt preparations will require use of standardized and clinically relevant in vitro assay systems and in vivo animal models for testing, as well as development of new outcome measures for inflammation and pain pathways in human OA.

Anti-apoptotic treatments prevent cartilage degradation after acute trauma to human ankle cartilage.

OBJECTIVES: To investigate the effect of anti-apoptotic agents on cartilage degradation after a single impact to ankle cartilage. DESIGN: Ten human normal tali were impacted with the impulse of 1 Ns generating peak forces in the range of 600 N using a 4 mm diameter indenter. Eight millimeter cartilage plugs containing the 4 mm diameter impacted core and a 4 mm adjacent ring were removed and cultured with or without P188 surfactant (8 mg/ml), caspase-3 (10 uM), or caspase-9 (2 uM) inhibitors for 48 h. Results were assessed in the superficial and middle-deep layers immediately after injury at day 0 and at 2, 7 and 14 days after injury by live/dead cell and Tunel assays and by histology with Safranin O/fast green staining. RESULTS: A single impact to human articular cartilage ex vivo resulted in cell death, cartilage degeneration, and radial progression of apoptosis to the areas immediately adjacent to the impact. The P188 was more effective in preventing cell death than the inhibitors of caspases. It reduced cell death by more than 2-fold (P<0.05) in the core and by about 30% in the ring in comparison with the impacted untreated control at all time points. P188 also prevented radial expansion of apoptosis in the ring region especially in the first 7 days post-impaction (7.5% Tunel-positive cells vs 46% in the untreated control; P<0.01). Inhibitors of caspase-3 or -9 were effective in reducing cell death in the impacted core only at early time points, but were ineffective in doing so in the ring. Mankin score was significantly improved in the P188 and caspase-3 treated groups. CONCLUSIONS: Early intervention with the P188 and caspase-3 inhibitor may have therapeutic potential in the treatment of cartilage defects immediately after injury.

Oral glucosamine modulates the response of the liver and lymphocytes of the mesenteric lymph nodes in a papain-induced model of joint damage and repair.

OBJECTIVES: To assess whether glucosamine (GlcN), an oral supplement commonly taken to relieve the symptoms of osteoarthritis, modulates the immune and inflammatory responses to joint injury in organs proximal to GlcN absorption; namely, the liver and the gut-draining lymph nodes. METHOD: Using a papain-injected knee mouse model, standard histological methods were used to validate our model and document the impact of GlcN (100mg/kg/day) on groups of C57BL/6 mice (n=5). Circulating inflammatory cytokines were assessed by Luminex-based immunoassays and the relevance of this cytokine profile on proteoglycan biosynthesis evaluated using a patellar-cartilage assay. Real-time PCR was used to document the role of the liver in cytokine production. Finally, we appraised the activation of mesenteric lymph nodes (MLNs) lymphocytes by flow cytometry. RESULTS: Papain significantly degraded the proteoglycans in the injected knees by 2 days. Cartilage proteoglycan content was significantly higher in GlcN-treated, papain-injected knees at Day 14. The peak concentration of serum pro-inflammatory cytokines occurred earlier and decreased sooner in the injected, GlcN-supplemented mice; this trend was in agreement with the expression of these factors by the liver. GlcN did not alter the percentage of MLN populations but accelerated their activation. CONCLUSIONS: Oral GlcN alters the physiology of the liver and MLNs, which in turn, could indirectly alter the biology of the injured joint.

Effects of long-term estrogen replacement therapy on articular cartilage IGFBP-2, IGFBP-3, collagen and proteoglycan levels in ovariectomized cynomolgus monkeys.

OBJECTIVE: The purpose of this study was to determine the effects of long-term estrogen replacement therapy (ERT) on insulin-like growth factor binding protein (IGFBP)-2, IGFBP-3, collagen and proteoglycan levels in the articular cartilage of the knee joint in a well-characterized monkey model of naturally occurring osteoarthritis (OA). A secondary aim was to evaluate the effect of soy phytoestrogen treatment on these articular cartilage components. DESIGN: Monkeys were ovariectomized and given ERT, soy phytoestrogen treatment or no treatment (control) for 3 years. Ten animals were randomly selected from each of the three groups and the cartilage was dissected from the proximal tibia and distal femur of the knee. Levels of IGFBP-2, IGFBP-3, and total protein were measured in cartilage desorptions, and proteoglycan levels and collagen levels were measured in the cartilage tissue. Sections from the tibial plateau of the opposite knee were immunostained using antibodies directed against IGFBPs and evaluated subjectively. RESULTS: IGFBP-3 levels were significantly higher, and total protein levels were significantly lower in the cartilage desorption samples from the estrogen-treated animals compared to the control animals. There were no significant differences in IGFBP-2, collagen or proteoglycan levels between the estrogen-treated and control groups. Soy phytoestrogen treatment had no significant effect on the levels of any of the cartilage components that were measured. The staining patterns observed by immunohistochemistry suggested local production of IGFBP-2 and IGFBP-3 by articular cartilage chondrocytes. CONCLUSIONS: Long-term estrogen treatment results in increased IGFBP-3 levels in articular cartilage without a significant change in IGFBP-2, collagen or proteoglycan content, and IGFBP-3 appears to be synthesized by articular cartilage chondrocytes. Long-term soy phytoestrogen treatment did not have a statistically significant effect on the levels of IGFBP-2, IGFBP-3, collagen or proteoglycan.

A fibrin-based arterial media equivalent.

We report here, with respect to collagen production and the mechanical properties of a fibrin-based media equivalent (ME), on our efforts to optimize the culture conditions of neonatal SMCs entrapped in tubular fibrin gels. We examined several factors, including the concentration of fibrinolysis inhibitor, the cell source and initial number, the addition of TGF-beta and insulin to the culture medium, and the time in culture. We found that varying the concentration of epsilon-aminocaproic acid (ACA), an inhibitor of fibrinolysis, did not affect the collagen production, but that lower concentrations resulted in a compromised physical integrity of the ME. While use of neonatal SMCs yielded superior results over adult SMCs, a higher initial cell number did not improve results. The addition of 1 ng/mL of TGF-beta to the medium increased the collagen content fourfold and the ultimate tensile strength (UTS) and modulus approximately tenfold after 3 weeks, while the addition of both TGF-beta and insulin improved collagen content sixfold and UTS and modulus almost 20-fold. Additional TGF-beta (5 ng/mL) did not improve any of the properties measured, but additional time in culture did. Samples incubated for 6 weeks with TGF-beta and insulin contained about seven times the amount of collagen and had a three-times higher UTS and modulus than did samples incubated for only 3 weeks. When compared to collagen MEs, the fibrin MEs compacted to a greater extent and were both stronger and stiffer when cultured under the same conditions, having after 6 weeks a tensile modulus and ultimate tensile strength similar to those of rat abdominal aorta.

The role of disc cell heterogeneity in determining disc biochemistry: a speculation.

The nucleus pulposus is a key player in very early disc degeneration. In the young disc, by acting as a water-like fluid, as opposed to a solid, it resists compression and instantaneously distributes forces evenly in all directions to the inner annulus. The disc anlage notochordal cells contribute not only to how the disc develops, but also to the matrix of the young disc at a time when the nucleus is at its most fluid-like. In humans, the notochordal cells disappear early, when there is a transformation of the nucleus into a more solid cartilaginous tissue. In cell culture, the co-cultures of the notochordal cells and chondrocytic cells enhance proteoglycan synthesis by the opposite cell type due, at least partly, to soluble factors. The continued presence of notochordal cells in vivo may provide protection. In work by others, in vivo reinsertion of notochordal-rich nucleus pulposus in a damaged disc will delay annular degeneration. The notochordal cells in the nucleus may have a different phenotype from when they are in the notochord and they may go through a changing programme of expression critical to disc development and maintaining a fluid-like nucleus. Little is known about why, in many species, the notochordal cells die early during growth and only the chondrocytic cells persist. This area offers an interesting avenue of research that may lead to very early intervention in disc degeneration.

Enhanced fibrin remodeling in vitro with TGF-beta1, insulin and plasmin for improved tissue-equivalents.

The aim of this study was to better understand how to increase collagen content in and enhance mechanical properties of tissue-equivalents formed by entrapping tissue cells in fibrin gels, with the ultimate goal of developing mechanically robust artificial tissues. The two main areas of focus were cell culture medium composition and replacement frequency relative to a base case of incubating constructs in medium supplemented with just serum and replaced weekly. The optimal condition involved a combination of all three factors investigated-TGF-beta, insulin, plasmin-with medium replacement three times a week. Compared to a base case without these three factors, the combination case resulted in 20 times more collagen and a ten-fold increase in tensile strength. The high strain modulus and tensile strength were within an order of magnitude of cardiovascular tissues. This study indicates great potential for fibrin-based tissue-equivalents in soft connective tissue applications.

Fibrin as an alternative biopolymer to type-I collagen for the fabrication of a media equivalent.

We report here on studies examining the use of fibrin as an alternative to collagen for the entrapment of neonatal aortic rat smooth muscle cells (SMCs) in the fabrication of media equivalents. The studies show increased collagen production by fibroblasts entrapped in fibrin, which suggests that fibrin may be used in the fabrication of tissue equivalents to promote increased protein synthesis and remodeling. However, one of the challenges of working with fibrin is the rapid degradation by SMCs. This degradation was effectively inhibited with the addition of epsilon-aminocaproic acid (EACA) to the culture medium in concentrations ranging from 0.25 to 1 mg/mL. We also present results showing that fibrin stimulates collagen production by SMCs. SMCs in fibrin produced 3.2 and 4.9 times the amount of collagen produced by SMCs in collagen when supplemented with 1 and 0.25 mg/mL EACA, respectively. More than half of the collagen produced appeared in the medium rather than the matrix. The collagen in the medium appeared to be processed beyond the proform and may be in an aggregate form. In addition, the presence of type-III collagen or a type-I trimer was indicated by the results of an analysis of the medium by autoradiography.

Beta 1-integrins regulate the formation and adhesion of ovarian carcinoma multicellular spheroids.

Ovarian carcinoma multicellular spheroids are an in vitro model of micrometastasis whose adhesive abilities have not been elucidated. In this study, we identified adhesion molecules that mediate the formation of ovarian carcinoma spheroids and their subsequent adhesion to extracellular matrix proteins. The NIH:OVCAR5, but not the SKOV3, ovarian carcinoma cell line formed spheroids similar to multicellular aggregates isolated from patient ascitic fluid. NIH:OVCAR5 spheroid formation was augmented by a beta 1-integrin-stimulating monoclonal antibody or exogenous fibronectin, but was inhibited by blocking monoclonal antibodies against the alpha 5- or beta 1-integrin subunits. By immunohistochemical staining, alpha 2-, alpha 3-, alpha 5-, alpha 6-, and beta 1-integrin subunits, CD44, and fibronectin were detected in NIH:OVCAR5 spheroids. NIH:OVCAR5 spheroids adhered to fibronectin, laminin, and type IV collagen, and this adhesion was partially inhibited by blocking antibodies against the alpha 5-, alpha 6-, and alpha 2-integrin subunits, respectively. A blocking monoclonal antibody against the beta 1-integrin subunit completely inhibited adhesion of the spheroids to all three proteins. These results suggest that interactions between the alpha 5 beta 1-integrin and fibronectin mediate the formation of ovarian carcinoma spheroids and that their adhesion to extracellular matrix proteins at sites of secondary tumor growth may be mediated by a complex interaction between multiple integrins and their ligands.

Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells.

Bone marrow is the primary site of metastasis in patients with advanced stage prostate cancer. Prostate carcinoma cells metastasizing to bone must initially adhere to endothelial cells in the bone marrow sinusoids. In this report, we have modeled that interaction in vitro using two bone marrow endothelial cell (BMEC) lines and four prostate adenocarcinoma cell lines to investigate the adhesion mechanism. Highly metastatic PC3 and PC3M-LN4 cells were found to adhere rapidly and specifically (70-90%) to BMEC-1 and trHBMEC bone marrow endothelial cells, but not to human umbilical vein endothelial cells (15-25%). Specific adhesion to BMEC-1 and trHBMEC was dependent upon the presence of a hyaluronan (HA) pericellular matrix assembled on the prostate carcinoma cells. DU145 and LNCaP cells were only weakly adherent and retained no cell surface HA. Maximal BMEC adhesion and HA encapsulation were associated with high levels of HA synthesis by the prostate carcinoma cells. Up-regulation of HA synthase isoforms Has2 and Has3 relative to levels expressed by normal prostate corresponded to elevated HA synthesis and avid BMEC adhesion. These results support a model in which tumor cells with up-regulated HA synthase expression assemble a cell surface hyaluronan matrix that promotes adhesion to bone marrow endothelial cells. This interaction could contribute to preferential bone metastasis by prostate carcinoma cells.

Fibronectin and its fragments increase with degeneration in the human intervertebral disc.

STUDY DESIGN: This laboratory-based experiment correlates fibronectin content of intervertebral disc with a morphologic grade of degeneration. OBJECTIVES: To correlate the fibronectin content of the anulus fibrosus and nucleus pulposus with a gross morphologic grade of disc degeneration, and to determine the molecular size of the extractable fibronectin. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration increases with age and can lead to low back pain. Fibronectin helps to organize the extracellular matrix and provides environmental cues by interaction with cell surface integrins. In other tissues, its synthesis is elevated in response to injury. Fibronectin fragments can stimulate cells to produce metalloproteases and cytokines and inhibit matrix synthesis. METHODS: In this study, 17 anuli fibrosis and 18 nuclei pulposus from 11 spines were graded by Thompson's gross morphologic scale. Fibronectin was sequentially extracted with 4 mol/L guanidine hydrochloride and trypsin, and then quantitated by enzyme-linked immunoassay. The size of extractable fibronectin was determined by Western blot analyses. RESULTS: The fibronectin content of the disc increased with grade and was significantly elevated between Grades 3 and 4. The percentage of extractable fibronectin varied widely, but it was more extractable from the nucleus. In both the nucleus and anulus, 30% to 40% of the extractable fibronectin existed as fragments. Many of the fragments contained functional heparin or collagen-binding sites. CONCLUSIONS: Fibronectin is elevated in degenerated discs and frequently present as fragments. Elevated levels of fibronectin suggest that disc cells are responding to the altered environment. Fibronectin fragments resulting from normal or enhanced proteolytic activity could be a mechanism that induces the cell to degrade the matrix further.

Mechanisms of stiffening and strengthening in media-equivalents fabricated using glycation.

We have recently reported that glycation can be exploited to increase the circumferential tensile stiffness and ultimate tensile strength of media-equivalents (MEs) and increase their resistance to collagenolytic degradation, all without loss of cell viability (Girton et al., 1999). The glycated MEs were fabricated by entrapping high passage adult rat aorta SMCs in collagen gel made from pepsin-digested bovine dermal collagen, and incubated for up to 10 weeks in complete medium with 30 mM ribose added. We report here on experiments showing that ME compaction due to traction exerted by the SMCs with consequent alignment of collagen fibrils was necessary to realize the glycation-mediated stiffening and strengthening, but that synthesis of extracellular matrix constituents by these cells likely contributed little, even when 50 micrograms/ml ascorbate was added to the medium. These glycated MEs exhibited a compliance similar to arteries, but possessed less tensile strength and much less burst strength. MEs fabricated with low rather than high passage adult rat aorta SMCs possessed almost ten times greater tensile strength, suggesting that alternative SMCs sources and biopolymer gels may yield sufficient strength by compositional remodeling prior to implantation in addition to the structural remodeling (i.e., circumferential alignment) already obtained.

A simple cryopreservation method for the maintenance of cell viability and mechanical integrity of a cultured cartilage analog.

A method for cryopreserving a 100-microm-thick sheet of tissue produced by cultured rabbit chondrocytes has been developed. The method maintains cell viability and avoids tissue fracture and degradation of mechanical properties. A slow-freeze, fast-thaw procedure with 2 M Me(2)SO as the cryoprotectant resulted in no tissue fracture and approximately 90% viable cells after storage in culture flasks at -80 degrees C. The cells in the retrieved tissue remained responsive to IL-1beta, and tensile and fracture toughness properties of the tissue were not degraded by cryopreservation.

Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins.

We have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell "niches" in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1alpha or PF4 (LTC-IC maintenance without GAGs, 32 +/- 2%; with GAGs, 95 +/- 7%; P <.001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1alpha, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 +/- 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1alpha. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1alpha, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)

Exploiting glycation to stiffen and strengthen tissue equivalents for tissue engineering.

Glycation, the nonenzymatic crosslinking of proteins by reducing sugars, is known to cause stiffening of soft tissues over a lifetime, particularly in diabetics. We show here that glycation due to elevated glucose and ribose concentrations in cell culture medium can be exploited in a matter of a few weeks of incubation to stiffen and strengthen tissue equivalents and to increase their resistance to collagenolytic degradation, all without loss of cell viability. Glycated tissue equivalents did not elicit inflammation or induce calcification upon subcutaneous implantation; rather, they were permissive to host integration and remodeling. Thus a pathological process might be used in a targeted way in tissue engineering to fabricate tissue equivalents with the required mechanical properties and desired resorption rate upon implantation.

Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis.

The disappearance of notochordal cells is correlated with early degenerative changes in the intervertebral disc. With increased disc degeneration there is a marked decrease in proteoglycan synthesis, resulting in loss of mechanical function. One possible mechanism for the decrease in proteoglycan synthesis is the loss of notochordal cells from the tissue. In this study, nucleus pulposus cells cocultured with notochordal cells exhibit an increase in proteoglycan synthesis. Interestingly, purified notochordal cells synthesize little proteoglycan as observed by [35S]sulfate incorporation into proteoglycans. The observed increase in proteoglycan synthesis does not appear to be dependent on cell-cell contact; rather it is the result of soluble factor(s) produced by notochordal cells. Finally, no difference in chondroitin sulfate chain size in notochordal-stimulated nucleus pulposus cells was observed which is consistent with an up-regulation in aggrecan core protein synthesis. These results are consistent with canine breeds where notochordal cells persist into adult age and disc degeneration is not observed. This suggests notochordal cells play a vital role in maintaining disc integrity.

Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche.

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P <.005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34(+)/HLA-DR- cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34(+) cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

Chondrocytes in culture produce a mechanically functional tissue.

A mechanically testable tissue was grown in vitro from rabbit chondrocytes that were initially plated at high density (approximately 80,000 cells/cm2). The DNA, collagen, and proteoglycan content, as well as the tissue thickness, tensile stiffness, and synthesis rates, were measured at 4, 6, and 8 weeks. The biochemical properties were similar to those for immature cartilage, with predominantly type-II collagen produced; this indicated that the cells retained their chondrocytic phenotype. The tissue formed a coherent mechanical layer with testable tensile stiffness as early as 4 weeks. The tensile elastic modulus reached 1.3 MPa at 8 weeks, which is in the range of values for native cartilage from the midzone. Collagen density was approximately 24 mg/ml at 8 weeks, which is about one-half the value for native cartilage, and the collagen fibril diameters were smaller. Chondrocytes in culture responded to culture conditions and were stimulated by cytokine interleukin-1beta. When culture conditions were varied to RPMI nutrient medium with lower fetal bovine serum and higher ascorbic acid concentrations, the thickness decreased and the modulus increased significantly. Interleukin-1beta, added to the 8-week culture for 2 weeks, caused a decrease of 60% in thickness, a decrease of 81% in proteoglycan content, and a decrease of 31% in collagen content; this is similar to the response of cartilage explants to interleukin-1beta. This cartilage analog may be useful as a model system to study structure-function relationships in cartilage or as cartilage-replacement tissue.

A role of chondroitin sulfate glycosaminoglycan binding site in alpha4beta1 integrin-mediated melanoma cell adhesion.

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.

The interaction of the zone of calcified cartilage and subchondral bone in osteoarthritis.

The zone of calcified cartilage (ZCC) forms an important interface between cartilage and bone for transmitting force, attaching cartilage to bone, and limiting diffusion from bone to the deeper layers of cartilage. The height of the ZCC is a relatively constant percent of articular cartilage and the height is maintained by a balance between progression of the tidemark into the unmineralized cartilage and changing into bone by vascular invasion and bony remodeling. During its formation, the cells that form the ZCC have properties similar to the cells of the growth plate. In the adult, the ZCC becomes quiescent but not inactive. The ZCC may be reactivated in osteoarthritis and may progressively calcify the unmineralized cartilage. This might contribute to cartilage thinning which would increase the concentration of forces across the uncalcified cartilage leading to more damage. Although the subchondral bony plate remodels extensively in osteoarthritis, there is little evidence that a change in the biomechanics of the plate directly initiates the osteoarthritic process in cartilage. However, increased repair by endochondral ossification of vertical cracks in the ZCC that penetrate into the marrow space could contribute to progression via changes in the ZCC.