Eugene J.-M. Thonar

Recombinant osteogenic protein-1 upregulates extracellular matrix metabolism by rabbit annulus fibrosus and nucleus pulposus cells cultured in alginate beads.

This study was performed to determine if recombinant human osteogenic protein‐1 (rhOP‐1) is effective in promoting matrix synthesis and matrix formation by rabbit nucleus pulposus (NP) and annulus fibrosus (AF) cells cultured in alginate beads. The effects of culturing the cells in the presence of various concentrations of rhOP‐1 were assessed by measuring changes in cell proliferation, proteoglycan (PG) and collagen synthesis and mRNA expression, and in the matrix contents of PG and collagen, as indicators of matrix accumulation. At high concentrations, rhOP‐1 had a moderate mitogenic effect on both NP and AF cells. It also stimulated the synthesis of PG and collagen in a dose‐dependent manner: this was associated with a corresponding increase in the expression of mRNA for aggrecan core protein and collagen type II. The stimulatory effect of rhOP‐1 on PG synthesis was more pronounced than that on collagen synthesis. Continuous treatment with rhOP‐1 led to an increase in the total DNA, PG and collagen contents in both NP and AF cultures. The results presented here provide evidence of the ability of rhOP‐1 to stimulate the metabolism of both rabbit AF and NP cells cultured in alginate beads.

Tumor invasion and host extracellular matrix

SummaryIn this review some of the major mechanistic pathways by which tumor cells are thought to invade host tissues are discussed. Tumor invasion has been conceived to be the result of pathological, close-range interactions between malignant cells and host stroma. The sequence of events that characterize invasion can be summarized as follows: (a) Tumor cell clusters break from the confinement of the primary tumor. Loss of intercellular junctions (desmosomes), alterations in the chemical composition and physical properties of the cell surface coat (loss of fibronectin and heparan sulfate; excessive amounts of hyaluronate), and loosening of cell-substrate interactions (loss of hemidesmosomes, fibronectin, and heparan sulfate), are among the most frequently listed causes of tumor cell shedding. (b) Increased proteolytic activities at the invasion front cause focal alterations in the surrounding extracellular matrix, thereby changing its physical properties. Collagenases and cathepsins, as well as elastase and other neutral proteinases are the enzymes most frequently associated with matrix destruction and invasion. In some tissues this process is effectively regulated by inhibitors of matrix-degrading, proteolytic enzymes. (c) Tumor cells migrate into the altered matrix, possibly moving as aggregates along guidance tracks provided by host structures (blood vessels, lymphatics, nerves) or matrix macromolecules (collagen and fibronectin tracks). Migration seems to be preceded by increased swelling of glycosaminoglycan (i.e., hyaluronate) in the matrix, ahead of the migrating cell population. Various host cell types (mast cells, fibroblasts, endothelial cells, macrophages, etc.) may participate in these events.

Synovial fluid levels of tumor necrosis factor alpha and oncostatin M correlate with levels of markers of the degradation of crosslinked collagen and cartilage aggrecan in rheumatoid arthritis but not in osteoarthritis.

OBJECTIVE To compare synovial fluid (SF) levels of oncostatin M (OSM), tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) in patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to determine which correlate best with SF levels of antigenic keratan sulfate (Ag KS), a marker of aggrecan catabolism, and pyridinium crosslinks, markers of the degradation of mature collagen molecules. METHODS SF was drawn from the knee joints of patients with RA (n = 31) or OA (n = 31). Levels of Ag KS, D-pyridinoline (D-Pyr), pyridinoline (Pyr), OSM, TNFalpha, and IL-6 were measured by enzyme-linked immunosorbent assay. RESULTS RA patients had higher median SF levels of OSM, TNFalpha, IL-6, and Pyr, but a lower median level of D-Pyr, than OA patients. In both groups, IL-6 levels correlated positively with those of OSM and TNFalpha. However, the correlation between levels of OSM and TNFalpha was only significant in the RA group. Ag KS and Pyr levels correlated positively in RA but not in OA. The correlation between TNFalpha and Ag KS was positive in RA and negative in OA. Further, in RA, OSM and IL-6 levels correlated strongly with Pyr and Ag KS levels but not with D-Pyr levels, while there were no strong correlations in OA for OSM or IL-6 levels with Pyr, Ag Ks, or D-Pyr levels. CONCLUSION This in vivo study suggests that TNFalpha and other proinflammatory cytokines are involved in the up-regulation of the coordinated degradation of cartilage aggrecan and collagen in RA. Further, OSM may act synergistically with other proinflammatory cytokines in up-regulating the production of metalloproteinases by chondrocytes in rheumatoid joints.

Serum levels of hyaluronan, antigenic keratan sulfate, matrix metalloproteinase 3, and tissue inhibitor of metalloproteinases 1 change predictably in rheumatoid arthritis patients who have begun activity after a night of bed rest.

OBJECTIVE To evaluate whether and how moderate physical activity following a night of rest influences serum levels of matrix metalloproteinase 3 (MMP-3), tissue inhibitor of metalloproteinases 1 (TIMP-1), antigenic keratan sulfate (Ag KS), and hyaluronan (HA) in 10 normal subjects and 38 patients with rheumatoid arthritis (RA). METHODS Blood was obtained from 20 RA patients before they arose from a nights sleep, and again 1 and 4 hours after they had begun to perform moderate physical activity. Another 18 RA patients remained in bed and blood was sampled at the same time periods. Serum levels of MMP-3, TIMP-1, Ag KS, and HA were measured by enzyme-linked immunosorbent assay. Clinical activity was evaluated by the Lansbury index. RESULTS Both in normal subjects and in RA patients who did not remain in bed throughout the period of blood sampling, levels of HA, Ag KS, and MMP-3 increased significantly during the first hour after the subjects arose: the increase in HA and Ag KS correlated with the Lansbury index in the RA group. Three hours later, levels of Ag KS had dropped to baseline values in both groups of subjects. Levels of HA remained significantly and moderately elevated in the RA group but not in the control group, while levels of MMP-3 did not drop significantly in either group. In contrast, levels of HA, Ag KS, and MMP-3 did not change significantly in RA patients who had remained in bed. Unlike the other markers, the levels of TIMP-1 remained unchanged at the different time periods in all 3 groups studied. CONCLUSION Significant changes in serum levels of some metabolic markers occur during the first hour after one arises from a night of sleep, especially in patients with RA. Measurement of the magnitude of these changes at different times in individual patients provides very different information about metabolic changes occurring in joint tissue than does measurement of the level of the markers at a single time point, as is usually currently reported.

Basic fibroblast growth factor accelerates matrix degradation via a neuro-endocrine pathway in human adult articular chondrocytes

Pain‐related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P‐induced inflammatory processes are mediated via signaling through a G‐protein‐coupled receptor, that is, neurokinin‐1 tachykinin receptor (NK1‐R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF‐2) or IL‐1β accelerate matrix degradation via a neural pathway upregulation of substance P and NK1‐R. We show here that substance P stimulates the production of cartilage‐degrading enzymes, such as matrix metalloproteinase‐13 (MMP‐13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK1‐R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein‐7, suggesting the dual role of substance P as both a pro‐catabolic and anti‐anabolic mediator of cartilage homeostasis. We report that bFGF‐mediated stimulation of substance P and its receptor NK1‐R is, in part, through an IL‐1β‐dependent pathway. J. Cell. Physiol. 215: 452–463, 2008.

Comparative effects of bone morphogenetic proteins and Sox9 overexpression on matrix accumulation by bovine anulus fibrosus cells: implications for anular repair.

Study Design. An in vitro biologic study comparing the effects of a series of bone morphogenetic proteins (BMPs) and Sox9 on the extracellular matrix accumulation by bovine anulus fibrosus (AF) cells. Objective. To compare the effects of adenoviral-mediated overexpression of various BMPs and Sox9 on extracellular matrix accumulation by AF cells in vitro. Summary of Background Data. Repair of the disrupted AF, which is perceived to be a potential therapy to diminish nucleus pulposus (NP) herniation, may also offer a treatment strategy for severe symptomatic degenerative disc disease. To date, no systematic comparison of a large group of growth factors in the AF has been published. In this study, we compared the effects of the adenoviral-mediated overexpression of 12 BMPs and Sox9 on extracellular matrix production by AF cells. Methods. Adult monolayer-cultured bovine AF cells were transduced with adenoviral vectors containing human BMP and green fluorescence protein (GFP) genes (AdBMPs), or Sox9 and GFP genes (AdSox9), or GFP gene alone (AdGFP, as negative control). Proteoglycan and collagen accumulation, and cell proliferation were measured for each of the treatment groups 6 days after viral transduction. Results. AF cells transduced with BMP-2, -3, -5, -7, -8, -12, -13, -14, and -15, and Sox9 accumulated significantly more collagen than AF cells transduced with AdGFP (control). AF cells transduced with AdBMP-2, -4, -7, -10, -12, and -13, and AdSox9 accumulated significantly more proteoglycans than AF cells transduced with AdGFP. Conclusion. We have demonstrated the relative effectiveness of 12 different BMPs and Sox9 on the stimulation of proteoglycan and/or collagen accumulation by AF cells. This study is the first to compare the relative effectiveness of various BMPs and Sox9 on extracellular matrix accumulation by AF. This information should prove useful to those seeking to develop a strategy for repair of the AF in humans.

Age-related differences in the accumulation and size of hyaluronan in alginate culture

The alginate bead culture system has unique properties that make it possible to study the accumulation and turnover of macromolecules in two distinct matrix compartments of the cartilage matrix: the cell-associated matrix (CM) and the further removed matrix (FRM). Taking advantage of this culture system, the purpose of this study was to examine age-related changes in the metabolism of hyaluronan (HA) in these two compartments. Bovine chondrocytes, isolated from fetal, young adult, and old adult articular cartilage, were cultured in alginate beads. On Days 7 and 14 of culture, the alginate gel was solubilized, the CM and FRM were separated and macromolecules in both compartments were analyzed. When compared to the cells from fetal and old adult animals, the young adult cells proliferated at the fastest rate. Fetal cells produced a more abundant CM that was richer in proteoglycans (PGs) than the CM of young or old adult cells. With increasing age, there was an increased tendency for PG, collagen, and HA to escape incorporation into the CM and to become immobilized in the FRM. Very striking changes also were observed in the ratio of HA to PG, which increased markedly with age, and in the size of the HA molecules, which decreased markedly with age. The results suggest that the metabolism of HA in cartilage undergoes pronounced age-related changes, some of which are retained during culture in alginate gel. The findings also suggest that the previously documented age-related decrease in the size of HA in native bovine cartilage reflects, at least in part, a biochemical process occurring at the time or at least soon after the glycosaminoglycan chain is synthesized. It does not appear to simply be the result of age-related changes occurring slowly with time after synthesis, as was previously suggested to be the case for human articular cartilage.

Osteogenic protein 1 in synovial fluid from patients with rheumatoid arthritis or osteoarthritis: relationship with disease and levels of hyaluronan and antigenic keratan sulfate.

The measurement of body fluid levels of biochemical markers in joint tissues has begun to provide clinically useful information. Synovial fluid (SF) plays an important role in articular joint lubrication, nutrition, and metabolism of cartilage and other connective tissues within the joint. The purpose of our study was to identify and characterize osteogenic protein 1 (OP-1) in SF from patients with rheumatoid arthritis (RA) or with osteoarthritis (OA) and to correlate levels of OP-1 with those of hyaluronan (HA) and antigenic keratan sulfate (AgKS). SF was aspirated from the knees of patients with either RA or OA and from the knees of asymptomatic organ donors with no documented history of joint disease. The presence of detectable OP-1 in SF was demonstrated by western blots with specific anti-pro-OP-1 and anti-mature OP-1 antibodies. Measurement of levels of OP-1, HA and AgKS was performed using ELISAs. OP-1 was identified in human SF in two forms, pro-OP-1 and active (mature) OP-1 – mature OP-1 being detected only in SF from OA patients and RA patients. Levels of OP-1 and HA were higher in RA patients than in OA patients and asymptomatic donors, while the level of AgKS was highest in SF from asymptomatic donors. Statistically significant differences were found between SF levels of OP-1 in RA and OA patients and between SF levels of AgKS among the three groups tested. The SF content of OP-1 tended to correlate positively with HA levels, but negatively with AgKS concentrations. In conclusion, the results of this study suggest that measurement of OP-1 in joint fluid may have value in the clinical evaluation of joint disease processes.

Proteoglycans nondissociatively extracted from different zones of canine normal articular cartilage : variations in the sedimentation profile of aggregates with degree of physiological stress

Proteoglycans were extracted and purified without dissociation (a-A1 preparations) from superficial and deeper layers of high weight-bearing (HWA) and low weight-bearing (LWA) areas of dog normal articular cartilage. These proteoglycans were then characterized by velocity gradient centrifugation. In each of the 4 different topographical regions, the weight average sedimentation coefficients related strongly with total hexuronate content of the tissue. In the superficial layers, almost all aggregates had low sedimentation coefficients: the aggregates were smaller and less abundant in LWA than in HWA. The deeper layers contained an additional population of faster sedimenting aggregates which appeared smaller and less abundant in LWA than in HWA. Quantification and functional characterization of aggregates as well as in vitro aggregating studies showed that the topographical differences in size and content of aggregates were related to differences in content of hyaluronate and link protein in the a-A1 preparations. Superficial a-A1 specimens contained twice as much hyaluronate as deeper a-A1 preparations and their hyaluronate content increased with degree of physiological stress. Deeper a-A1 specimens from weight-bearing areas did not differ in their hyaluronate content but experiments assessing the saturation with link protein of these different a-A1 preparations suggested that specimens from HWA contained more active link than those from LWA. In contrast, the capacity of aggregation of a-A1D1D1 proteoglycan monomers as well as the molecular weight (Mr = 5 x 10(5) and aggregating capacity of hyluronate molecules appeared very similar in all a-A1 preparations from areas of articular cartilage. It is hypothesized that the synthesis of the three constituents necessary for aggregate formation (i.e. proteoglycan monomers as well as hyaluronate and link protein molecules) increases with degree of physiological load and that aggregation helps to maintain within cartilage the high concentration of proteoglycans that are essential for its biomechanical functions. The reported topographical variations in the distribution of proteoglycan aggregates reflect probably a maximal adaptation of the physiologic and biomechanical properties of the matrix to meet the high stress levels experienced by the articular cartilage in vivo.

CHAPTER 25 – Products of Cartilage Metabolism

This chapter discusses the major products of articular cartilage metabolism with special emphasis on those that are relevant as body fluid markers. It provides a brief review of the structure–function relationships of the major matrix building blocks. The cartilage matrix contains several molecules that are found in much higher concentrations than in other tissues. The attention that aggrecan and collagen type II are currently receiving as potential markers of specific metabolic alterations in cartilage disease will likely extend to less abundant molecules found to play key roles in the maintenance of matrix homeostasis. As the understanding of the function and metabolism of these molecules improves, so will their potential as markers of the irreversible destabilization of the collagenous meshwork or of other specific alterations in cartilage matrix organization.