Extracellular superoxide dismutase and oxidant damage in osteoarthritis

Abstract

Objective: To use human cartilage samples and a mouse model of osteoarthritis (OA) to determine whether extracellular superoxide dismutase (EC-SOD) is a constituent of cartilage and to evaluate whether there is a relationship between EC-SOD deficiency and OA.

Methods: Samples of human cartilage were obtained from femoral heads at the time of joint replacement surgery for OA or femoral neck fracture. Samples of mouse tibial cartilage obtained from STR/ort mice and CBA control mice were compared at 5, 15, and 35 weeks of age. EC-SOD was measured by enzyme-linked immunosorbent assay, Western blotting, and immunohistochemistry techniques. Real-time quantitative reverse transcription-polymerase chain reaction was used to measure messenger RNA for EC-SOD and for endothelial cell, neuronal, and inducible nitric oxide synthases. Nitrotyrosine formation was assayed by Western blotting in mouse cartilage and by fluorescence immunohistochemistry in human cartilage.

Results: Human articular cartilage contained large amounts of EC-SOD (mean +/- SEM 18.8 +/- 3.8 ng/gm wet weight of cartilage). Cartilage from patients with OA had an approximately 4-fold lower level of EC-SOD compared with cartilage from patients with hip fracture. Young STR/ort mice had decreased levels of EC-SOD in tibial cartilage before histologic evidence of disease occurred, as well as significantly more nitrotyrosine formation at all ages studied.

Conclusion: EC-SOD, the major scavenger of reactive oxygen species in extracellular spaces, is decreased in humans with OA and in an animal model of OA. Our findings suggest that inadequate control of reactive oxygen species plays a role in the pathophysiology of OA.

Figure 1

Extracellular superoxide dismutase (EC-SOD) in normal and osteoarthritic (OA) human cartilage and in human lung tissues. A, Baseline data in the OA and femoral neck fracture (normal control) patients. There was a preponderance of women in both groups. The control group was slightly older than the OA group and had a trend toward a higher body mass index (BMI). Linear regression modeling showed that only disease state (OA or normal control) was a significant predictor of the EC-SOD level. B, Levels of EC-SOD in lung tissue from cadaver donors and in normal articular cartilage from patients with femoral neck fracture, as determined by enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody against recombinant human EC-SOD. Cartilage, with its extensive extracellular matrix, has large amounts of EC-SOD compared with lung tissue. C, Levels of EC-SOD per gram of cartilage wet weight in a subset of OA and normal control cartilage, as determined by ELISA. There is an ~4-fold decrease in EC-SOD content in OA cartilage compared with control cartilage. D, Levels of EC-SOD protein in a random subset of cartilage samples from OA patients and normal controls, as determined by ELISA. Again, there is an ~4-fold decrease in EC-SOD protein in OA cartilage compared with control cartilage. Except where indicated otherwise, values are the mean and SEM.

Figure 2

Immunolocalization of extracellular superoxide dismutase (EC-SOD) in normal and osteoarthritic (OA) human cartilage. Immunohistochemistry of EC-SOD in human cartilage samples was performed using polyclonal antibody to human EC-SOD and peroxidase staining. Negative controls were prepared with EC-SOD-absorbed antiserum. A, Cartilage section from a 37-year-old male cadaver donor, showing significant EC-SOD staining in the matrix surrounding the chondrocytes, with variable light staining intracellularly. B, Negative control for the section shown in A. C, Cartilage from a 58-year-old woman with OA, showing increased cellularity, degeneration of the superficial cartilage layer, and pronounced lack of matrix staining. Note the increased intracellular staining for EC-SOD compared with the nonarthritic chondrocytes. D, Negative control for the section shown in C. (Original magnification × 100.)

Figure 3

Messenger RNA for human extracellular superoxide dismutase (EC-SOD), inducible nitric oxide synthase (iNOS), neuronal NOS (nNOS), and endothelial cell NOS (eNOS) in human articular cartilage. RNA was extracted from OA (n = 36) and control (n = 16) cartilage, and quantitative real-time reverse transcription-polymerase chain reaction was used to measure levels of mRNA for human EC-SOD (hEC SOD) (A), iNOS (B), nNOS (C), and eNOS (data not shown). Values were normalized against 18S and expressed as a ratio to 18S. We did not detect eNOS in any of the cartilage samples. Levels of mRNA for EC-SOD (z = -2.11, P = 0.03) and nNOS (z = -2.2, P = 0.03) were significantly increased in OA patients compared with controls, whereas levels of mRNA for iNOS were increased 4.6-fold, but the increase was not statistically significant (P = 0.3), by Wilcoxon's rank sum test. Values are the mean and SEM.

Figure 4

Levels of extracellular superoxide dismutase (EC-SOD) and nitrotyrosine formation in the STR/ort mouse model of osteoarthritis (OA). Tibial plateau cartilage samples from STR/ort and control CBA mice ages 5, 15, and 25 weeks were compared for levels of EC-SOD protein and mRNA by Western blotting and reverse transcription-polymerase chain reaction techniques (RT-PCR), as well as for evidence of nitrotyrosine formation. A, EC-SOD protein expression (mean and SEM). At 5 weeks of age, there is a dramatic decrease in EC-SOD protein in cartilage from STR/ort mice compared with CBA mice, which do not develop arthritis. At this age, there is no histologic evidence of arthritis in STR/ort mice. * = t = 8.48, P < 5 × 10^-5. B, EC-SOD mRNA levels (mean and SEM), as measured by RT-PCR. At 5 weeks of age, there is a decrease in EC-SOD mRNA in cartilage from STR/ort mice compared with control mice, suggesting either a failure of transcription or a rapid turnover of the pool. * = t = 10.54, P < 0.001. C, Levels of extractable nitrotyrosinylated proteins (mean and SEM). There is a small, but significant, decrease in STR/ort mice compared with control mice at ages 5 weeks (* = t = 3.93, P < 0.001), with progressive increases over time (* = t = 5.4, P < 0.0009 at 15 weeks and * = t = 6.27, P < 0.0004 at 25 weeks). The progressive increase in nitrotyrosinylated proteins despite the apparent correction of EC-SOD protein and mRNA levels suggests ongoing oxidant stress in the tissue following the initial insult. The initial EC-SOD deficiency with oxidative damage at 5 weeks may compromise the function of key proteins or signaling pathways in chondrocytes or in the matrix, leading to more oxidative damage and arthritis. D, Representative Western blot of nitrotyrosinylated proteins in cartilage from 2 CBA mice and 2 STR/ort mice ages 25 weeks. At least 3 proteins with molecular weights of ~75, 66, and 60 kd, indicating nitrotyrosine formation, were dramatically increased in STR/ort cartilage compared with control cartilage.

Figure 5

Nitrotyrosine in the extracellular matrix (ECM) of human cartilage samples, as determined by fluorescence immunohistochemistry. A, Osteoarthritic cartilage immunostained for 3-nitrotyrosine shows a distinct speckled pattern of labeling in the pericellular ECM as well as intracellular fluorescence. B, Control cartilage shows intracellular staining for 3-nitrotyrosine, but no staining of the ECM. (Original magnification × 100.)