EC-SOD has not previously been studied in articular cartilage. We found that it was present in large amounts in normal human cartilage and was decreased ~4-fold in OA cartilage. Extracellular SOD is the major scavenger of superoxide in the extracellular spaces and fluids. It is highly expressed in blood vessels, especially arteries, and vascular smooth muscle, as well as in lung tissue. Both tissues have shown important correlations between EC-SOD deficiency and diseases (28
). Deficiency of EC-SOD increases bleomycin-induced pulmonary fibrosis (37
) and increases sensitivity to hyperoxic lung injury (38
). Two recent studies of the vascular system associated EC-SOD deficiency with cardiovascular disease (29
). Our findings that EC-SOD is present in 10-fold higher concentrations in articular cartilage as compared with lung tissue and that it is markedly decreased in OA cartilage as compared with normal cartilage suggest that EC-SOD plays a key role in modulating ROS in cartilage.
Because cartilage is avascular and hypoxic, the concept that ROS may play a part in tissue regulation is counterintuitive. Oxygen concentrations in cartilage have been shown to range from 5% at the surface to 1% in the deeper zones (40
). Chondrocytes do produce ROS when grown in culture at 21% oxygen and possess an NADPH oxidase-like complex (12
), but the effect on ROS production has not been studied under conditions of low oxygen pressure. However, hypoxia has been shown in other systems to induce a paradoxical increase in oxygen radicals, especially superoxide, apparently due to a shift to 1-electron transfers in the electron transport chain (41
). Identifying large amounts of EC-SOD, an important ROS scavenger, in cartilage tissue suggests that ROS scavenging is necessary for homeostasis and that ROS are produced in significant amounts.
Collagen is particularly vulnerable to oxidant damage, which impairs its mechanical properties (5
). Proteoglycans in the ECM or extracellular fluids can be cleaved by ROS (6
). Damage to these structural proteins from excess oxidants would leave the cartilage at risk of mechanical failure under normal loads and would change the water-holding capacity of the tissue. Decreased tensile strength is seen in OA cartilage in association with damage to collagen fibrils (43
). Interestingly, this damage to the collagen fibrils has been shown to originate around chondrocytes (44
). Since chondrocytes are the source of ROS, whether generated by mechanical forces or cytokines, the fact that the initial collagen damage is found in pericellular areas is consistent with a local pericellular deficiency of oxidant scavenging.
A superoxide molecule produced in the extracellular space by a membrane oxidase would potentially remain in the ECM until it is either scavenged or reacts with one of the molecules of the ECM, such as collagen or aggrecan. The diffusion path, or distance that the molecule can travel before reacting, has not been studied for superoxide in the ECM, but in the vascular system, the diffusion path was found to be ~40μ (45
). Superoxide is highly reactive with hemoglobin, and thus, the diffusion path is likely to be greater in avascular cartilage. The distance that a reactive molecule can travel in a tissue is related to its reduction potential and charge. Because of its high reactivity (reduction potential 2.31V), the hydroxyl radical would have a much shorter diffusion path than superoxide (reduction potential 0.94V). Since superoxide is negatively charged, it does not diffuse through plasma membranes.
The local pericellular loss of EC-SOD that we found in the OA cartilage, as shown in , could account in part for the pericellular collagen damage and the mechanical deficiency of the OA cartilage. Our observation that EC-SOD is decreased in OA cartilage may represent an important etiologic factor, representing either an initial trigger for the disease due to a congenital deficiency in EC-SOD or an accelerator of the process after some of the proteoglycans and their EC-SOD-binding sites are lost from the tissue.
Superoxide and nitric oxide react rapidly in biologic systems to produce toxic species, including peroxynitrite and the hydroxyl radical. With adequate local extracellular SOD, superoxide is removed from intercellular spaces and NO signaling can occur (46
). Because superoxide is formed in response to both inflammatory cytokines and damaging mechanical forces, the capacity to scavenge excess superoxide may represent an important control point for maintaining protection of the ECM structural molecules. Although low levels of hydrogen peroxide have been shown to stimulate synthesis and may help to repair matrix after mechanical injury (10
), with inadequate EC-SOD, the balance may shift to an oxidized/inflammatory state with formation of the more toxic radicals.
Del Carlo and Loeser (47
) have shown that NO alone is not sufficient to cause chondrocyte apoptosis, but that it requires the addition of more ROS. This finding correlates well with the dualistic effects of NO shown in other biologic systems where NO alone is a physiologic signaling molecule and the damaging effects require an oxidizing environment (48
). Excess ROS can result in damage to the structural proteins, collagen, and proteoglycans if no mechanism is present to scavenge the reactive molecules, particularly if the proinflammatory cytokines activate NF-κB, inducing iNOS expression and producing NO in association with superoxide (49
We found that EC-SOD mRNA was increased in OA cartilage in concert with the local deficiency of EC-SOD protein. We also found mRNA for both iNOS and nNOS were increased proportionately to the ECSOD mRNA in the OA cartilage (). In dermal fibroblasts, EC-SOD has been shown to be up-regulated by the inflammatory cytokines tumor necrosis factor α and interferon-γ (50
). EC-SOD was also shown to be temporally coregulated with iNOS in response to NF-κB activation (51
). Although the factors that regulate ECSOD expression in chondrocytes have not been defined, our findings are consistent with a coordinated response.
Our subject population represents the severe end of the OA disease spectrum, given that the disease had progressed to necessitate joint replacement. Since tissue samples were obtained late in the disease process, the findings may not represent the biochemical situation at disease onset. Although alterations in the redox state of the chondrocytes may constitute a part of the initiation or propagation of OA, the changes in EC-SOD in the tissue may also represent secondary effects of proteoglycan loss and lack of binding sites in the matrix. We therefore turned to an animal model of spontaneous OA to define whether there were changes in the oxidative state of cartilage early in the disease.
The STR/ort mouse model allowed us to study alterations in EC-SOD during the prelesional period, which is not possible to study in human OA. We found that the STR/ort mouse is deficient in EC-SOD weeks before developing histologic lesions, which suggests that inadequate scavenging of ROS occurs before the matrix is disrupted. The presumed increase in ROS due to EC-SOD deficiency is associated with a progressive increase in nitrotyrosine residues in the cartilage. The response of increased EC-SOD in the tibial cartilage was not adequate to prevent continued oxidative stress in the tissue, as demonstrated by the increasing nitrotyrosine formation at 15 and 25 weeks of age.
The fact that the EC-SOD level in the STR/ort mice increased to the level in the control CBA mice does not mean that it retained adequate scavenging capacity for tissue that had already sustained oxidative damage and had increased production of ROS. Studies in other systems have shown that when oxidative stress occurs, the response of the antioxidant enzymes is to rise to supranormal levels in order to control the excess ROS being generated. This supranormal increase in antioxidants did not occur in the STR/ort mice, and the state of oxidative stress was not corrected. Demonstrating deficiency of EC-SOD in the young mice in association with progressive increases in nitrotyrosine over the time course of the disease represents an important corroboration of the postulated role of ROS and EC-SOD deficiency in the initiation of OA.
We believe that the mouse cartilage, with lower levels of EC-SOD found in 5-week-old animals before the development of OA lesions, reflect an initiating event that causes oxidative stress/damage to key proteins in the murine joint. We postulate that this continuing oxidative damage, despite the reactive increase in ECSOD levels in the cartilage, acts in an unknown manner to trigger the progressive loss of cartilage. The reactive increase in EC-SOD in the STR/ort mouse cartilage is inadequate to manage the ROS that are being generated, even though the chondrocytes are able to respond in part to the oxidative stress.
In the cartilage samples from the OA patients, we are looking at the very late stages of the disease. Proteoglycan has been lost, although the tissue architecture is retained, and adjacent tissue has undergone complete destruction. Further study of human tissues is needed to define the disease process, but we would postulate that after a period of compensation for increased oxidative stress, there is a late stage in which the continued production of EC-SOD is inadequate and that this, coupled with losses of binding sites, leads to a late phase of low EC-SOD in the tissue.
EC-SOD is secreted with a positively charged binding site and binds to collagen and proteoglycans in the ECM. The high levels of EC-SOD that we found in normal cartilage may play several critical roles in the tissue, such as to protect the vulnerable structural proteins in the ECM from direct oxidant damage, to limit the formation of peroxynitrite and prevent its deleterious effects on proteins and the cytoskeleton, to permit the unopposed signaling of NO in the tissue, and to modulate the local redox state and signaling pathways in the pericellular matrix and chondrocytes.
Articular cartilage is a mechanically sensitive tissue in which alterations in mechanical loading affect the synthetic and degradative pathways, and chondrocytes are able to respond to harmful or beneficial forces with different cellular biochemical messages (52
). OA chondrocytes exposed to shear stress up-regulate iNOS in association with an inhibition of collagen and aggrecan synthesis, and both NO and superoxide are produced in cartilage tissue exposed to prolonged compression (16
). Both the mechanical messages and cytokine messages in cartilage appear to be mediated by NO and ROS. Since EC-SOD scavenges superoxide, it can modulate the signaling events on the cell surface between NO alone and within an oxidizing environment. Unopposed signaling of NO may reduce inflammation by inhibiting NF-κB (54
). Use of SOD mimetics has been shown to reduce the severity of inflammation in collagen-induced arthritis (55
OA is a multifactorial disease in which genetic influences and environmental factors affect the occurrence of the disease and the age at onset. Increased body weight, excessive shearing forces from a malaligned or unstable joint, or unusual loading from occupational activities might be transduced as increased reactive molecules in the cartilage, but the variation in scavenging capacity within the joint would account for the individual differences in the incidence of arthritis or the age at onset. The individual capacity to buffer ROS can be related to genetic factors, possibly affected by hormones, or decreased by other disease processes that generate excess ROS (e.g., iron overload). By demonstrating that cartilage contains large amounts of ECSOD, we propose that its role is to protect the extracellular structural proteins from ROS that is routinely generated in the tissue. EC-SOD deficiency may be involved in the disease process as a result of early matrix changes and loss of binding sites in the tissue or as a result of an inadequate production of the enzyme. In showing an association between OA and a decrease in a major extracellular ROS scavenger in cartilage, we suggest that damage from ROS plays a key role in either the initiation or the propagation of OA.
We propose that an altered redox state in articular cartilage can lead to some of the major findings that are associated with OA and offer a means of integrating the role of mechanical factors and the varying predisposition of individuals to the disease. In concert with the direct oxidant damage to the structural proteins, the additional effects of abnormal redox signaling may lead to a failure of the repair processes or to a decreased responsiveness to growth factors (56
). Our findings of profoundly decreased levels of EC-SOD in OA cartilage, both in humans and in an animal model of spontaneous OA, support our thesis and suggest that this is an important area for further research into new treatment options.