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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Objective

To investigate the role of oxidative functions in human osteoarthritic (OA) chondrocytes and to investigate the presence of in vivo molecular markers of lipoxidation in OA cartilage.

Methods

An in vitro model of cartilage collagen degradation was used. Lipid peroxidation activity and overall oxidative function in OA chondrocytes were monitored by cis-parinaric acid and dichlorofluorescein assays, respectively. In vivo molecular markers of lipoxidation in normal and OA cartilage were studied using immunohistochemistry to detect the presence of malondialdehyde and hydroxynonenal adducts.

Results

Human OA chondrocytes showed a robust amount of 3H-proline–labeled collagen degradation upon stimulation with lipopolysaccharide and calcium ionophore A21387, as compared with that in untreated OA chondrocytes. Primary OA chondrocytes showed both spontaneous and inducible levels of lipid peroxidation activity. However, lipid peroxidation activity was already maximally elevated in more than 50% of the OA chondrocyte samples. Overall, spontaneous and inducible oxidative activities were observed in all OA samples. Immunohistochemical analysis of control OA tissue sections that were not treated with monoclonal antibody showed little immunoreactivity. OA cartilage sections treated with monoclonal antibodies showed specific immunoreactivity on the cartilage surface, at sites of OA lesions, at the pericellular matrix, and at intra- and intercellular matrices. Normal cartilage sections showed faint surface reactivity.

Conclusion

Our observations suggest that human OA chondrocytes demonstrate spontaneous and inducible cell-associated lipoxidative and nonlipoxidative activity. Lipoxidative activity appears to be enhanced in OA chondrocytes. The presence of molecular markers of in vivo lipid peroxidation was demonstrated in OA cartilage, suggesting its role in the pathogenesis of the disease.

Osteoarthritis (OA) is the most common form of joint disease that affects humans. The incidence of OA increases during every decade of life, and by the age of 65 years, almost one-third of the population has OA of the knee joints. The economic burden attributed to the joint pain and disability of OA amounts to billions of dollars each year (1). As the population demographic in the US changes to a predominantly older generation, the increasing prevalence of OA will be a major public health problem.

There is currently no treatment available that will prevent or cure OA. Pharmacologic and nonpharmacologic agents used for OA provide only symptomatic relief of pain. The lack of specific therapy for this disease is perhaps due to our limited understanding of its pathogenesis. Understanding the molecular mechanisms involved in the development of OA will help us to develop ways to prevent or reverse the degenerative process of the disease.

Current concepts of the pathogenic mechanisms of OA suggest that there is a shift in the homeostatic balance between the destruction and synthesis of bone and cartilage, with a net progressive destruction of these tissues (2). The destructive process is mediated by the production of aggrecanases and matrix metalloproteases that selectively degrade cartilage matrix (3–5). In addition to the role of proteases in matrix destruction, recent studies of the biology of chondrocytes show that these cells actively produce reactive oxygen species (ROS), including superoxide anions, hydrogen peroxide, hydroxyl radicals, and nitric oxide (6–13). ROS are capable of inducing apoptotic cell death in chondrocytes, but more important, they can result in the degradation of aggrecan and collagen (14–20). Degradation of aggrecan and collagen by ROS has been studied in some detail (15–20). However, most of these studies have been performed in vitro using purified matrix components and ROS. The in vivo role of ROS in the matrix degradation of cartilage during aging and OA remains to be determined. Immunochemistry studies show the presence of 3-nitrotyrosine residues in human articular cartilage that correlate with aging and OA, which suggests that oxidative damage from reactive nitrite radicals may contribute to the development of age-related OA (21).

In other age-related degenerative diseases, such as those of the brain and cardiovascular systems, lipid peroxidation has been implicated as the key source of oxidative stress (22). It is possible that lipid radicals could also be a source of oxidative stress in age-related OA. Lipid peroxidation generates a variety of hydroperoxide and aldehyde products that are highly reactive with components of the cell and the extracellular matrix (23, 24). Lipid radicals also function as intracellular signaling molecules, influencing various cellular functions (25, 26). There is a paucity of knowledge about the role of lipid-induced oxidative stress in cartilage aging and OA.

We have shown that chondrocyte-derived ROS mediate aggrecan degradation and that various antioxidants prevent this degradation process (27). In ensuing studies, we investigated the role of chondrocyte-dependent ROS-mediated collagen degradation (28). For those investigations, we developed an in vitro model of chondrocyte-dependent collagen degradation. Using this model, we showed that chondrocyte-derived lipid peroxidation mediates collagen degradation and that vitamin E inhibits this degradation process (28). More recently, we demonstrated that malondialdehyde (MDA), a toxic aldehydic end product of lipid peroxidation, mediates the oxidation of cartilage collagen (29). MDA and hydroxynonenal (HNE) are specific and major aldehydic products of lipid peroxidation that are believed to be largely responsible for the cytopathologic effects observed during the oxidative stress of lipid peroxidation (23, 24). Taken together, these observations suggest a role of lipid peroxidation in cartilage aging and OA.

In the present study, we investigated the role of lipoxidation in collagen degradation by human articular chondrocytes. We also investigated the presence of in vivo molecular imprints of lipid peroxidation in human OA cartilage. Our findings are presented herein.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Reagents.

Lipopolysaccharide (LPS) from Escherichia coli 0127:B8, phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP), calcium ionophore A23187, concanavalin A (Con A), and vitamin E were purchased from Sigma (St. Louis, MO). Reagent-grade hydrogen peroxide was obtained from Fisher Scientific (Fair Lawn, NJ). Dulbecco's minimum essential medium (DMEM), fetal bovine serum (FBS), Hanks' balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), L-glutamine, gentamicin, HEPES buffer, penicillin, and streptomycin were purchased from Gibco (Grand Island, NY), and L-proline-(2,3-3H) with a specific activity of 1.6 GBq/mmole was obtained from DuPont NEN (Wilmington, DE).

Human cartilage samples.

Samples of OA cartilage from hip or knee joints and normal cartilage from finger or knee joints were obtained from patients undergoing surgery. Patients with OA were ages 55–75 years. Patients with normal cartilage were ages 35, 45, and 50 years. All cartilage samples were obtained in accordance with the approval of the Institutional Review Board.

Isolation of articular chondrocytes.

Chondrocytes were isolated as described previously (30). The viability of chondrocytes was confirmed by trypan blue exclusion. Primary chondrocytes were resuspended in 10% FBS in DMEM containing antibiotics (1%) and HEPES buffer (10 mM, pH 7.4) (complete medium). The cells were cultured in 100-mm petri dishes and multiwell plates.

Experimental design.

Primary human OA articular chondrocytes were distributed into 24-well plates at a concentration of 1–2 × 105 cells/well in 1 ml of complete medium (28). Chondrocytes were allowed to attach for 3–5 days, and the medium was changed every 3 days. Confluent cells in multiwell plates were labeled with 3H-proline (1–2.5 μCi/well) during the last 24–48 hours of culture. The cell monolayer was washed at least 4–5 times with warm HBSS by flipping the plates to remove unincorporated proline from the matrix. Albumin or serum-free EBSS was added to the wells. Experiments were performed in triplicate wells. The test reagents were then added, and the total volume was adjusted to 0.5 ml with EBSS. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 4–24 hours. Aliquots (100 ml) were removed and processed for scintillation counting. The plastic-bound 3H-proline–labeled matrix (the residuum) was solubilized with 0.5M NaOH and counted. The percentage of 3H-proline–label released was calculated, and these data are shown in the figures below.

Determination of lipid peroxidation.

Lipid peroxidation in chondrocytes was measured by the cis-parinaric acid method described by Hedley and Chow (31). Parinaric acid is a naturally fluorescent fatty acid that readily incorporates into cell membranes. Its loss of fluorescence over time has been used to monitor lipid peroxidation and to study the protective effect of antioxidants. Trypsin–EDTA–released confluent primary chondrocytes were loaded with 10 μM cis-parinaric acid for 1 hour at 37°C and washed. The fluorescence due to parinaric acid was monitored at 37°C using a Perkin Elmer LS-5B luminescence spectrometer (Perkin-Elmer Cetus, Norwalk, CT) set at 325 nm excitation and 405 nm emission.

Assay for 2′,7′-dichlorofluorescein diacetate (DCF-DA) oxidation.

Detached chondrocytes were incubated at 37°C with 5 μM DCF-DA for 60 minutes. Fluorometric detection of DCF oxidation was performed using a Perkin Elmer LS-5B luminescence spectrometer set at 488 nm excitation and 525 nm emission, using a 10-nm slit width (6).

Immunohistochemical analysis.

OA tissue samples were obtained from visible sites of OA lesions. Full-thickness tissue pieces minus the calcified cartilage were obtained from OA and normal cartilage, and the sections were aligned so that the top of the sections shown in the photomicrographs below represents the top surface of the cartilage. Normal and OA cartilage samples were mounted in OCT embedding medium (Miles, Naperville, IL), rapidly frozen, and 6-μm–thick sections were cut at −20°C using a TissueTek II cryostat. Sections were placed on glass microscope slides that had been precoated with aminoalkylsilane to ensure complete adherence. Sections were fixed for 5 minutes in 4% freshly prepared paraformaldehyde in phosphate buffered saline (PBS) followed by several washes with PBS. Nonspecific binding was blocked by horse serum in PBS. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide.

To enhance the permeability of the extracellular matrix, tissues were treated for 90 minutes at 37°C in a humidified chamber using chondroitinase ABC (Sigma) in PBS, pH 7.6, at a concentration of 0.0123 units/50 μl per section. Tissue sections were washed with 1% bovine serum albumin (BSA) in PBS. Consecutive tissue sections were either not treated with primary antibody or were treated with mouse monoclonal MDA2 (1:200 dilution in PBS with 1% BSA) or NA59 (1:20 dilution in PBS with 1% BSA) antibody for 45 minutes at room temperature in a humidified chamber. The antibodies MDA2, which is specific for MDA-modified lysine, and NA59, which is specific for 4-HNE–modified lysine, were kindly provided by Dr. W. Palinski (University of California, San Diego, CA) (32). The sections were washed 3 times with PBS 0.1% BSA.

For the secondary antibody, slides were treated for 30 minutes at room temperature with pig F(ab′)2 anti-mouse Ig labeled with biotin and peroxidase-conjugated streptavidin as indicators (American Corporation, Arlington Heights, IL). Slides were washed and developed with diaminobenzidine. Tissue sections were also stained with hematoxylin and eosin.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Induction of the release of 3H-proline–labeled cartilage collagen matrix by treatment with hydrogen peroxide, calcium ionophore, or LPS.

The susceptibility of 3H-proline–labeled collagen matrix to oxidant damage was investigated by exposing OA chondrocytes to a bolus (2 mM and 20 mM) of hydrogen peroxide. As shown in Figure 1, the release of 3H-proline–labeled collagen was significantly enhanced at 4 hours in cultures exposed to hydrogen peroxide. The release of labeled collagen by hydrogen peroxide was dose- and time-dependent (data not shown). Hydrogen peroxide has been implicated in aggrecan and collagen degradation (27, 28). The data indicate that the release of labeled matrix into the culture medium corresponds to the known oxidative damaging potential of hydrogen peroxide on the cartilage matrix.

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Figure 1. Induction of the release of 3H-proline–labeled collagen from osteoarthritic (OA) articular chondrocytes by lipopolysaccharide (LPS), calcium ionophore A23187, and hydrogen peroxide. 3H-proline–labeled monolayers of primary OA articular chondrocytes in 24-well plates were stimulated with the indicated concentrations of calcium ionophore A23187 (Ca Ion), LPS, concanavalin A (Con A), phorbol myristate acetate (PMA), and formyl-methionyl-leucyl-phenylalanine (FMLP) and the indicated boluses of hydrogen peroxide. Results from a representative experiment are shown. Values are the mean of triplicate wells. ∗ = P < 0.05 versus control, by Student's t-test.

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OA chondrocytes were also treated with a variety of agonists (LPS, PMA, FMLP, Con A, and A23187) that have been shown to induce oxidative burst activity in chondrocytes. Of the agonists tested, only calcium ionophore A23187 and LPS produced statistically significant increases (P < 0.05 by Student's t-test) in the release of labeled collagen matrix, as compared with the background release from untreated cells (Figure 1). Both LPS and A23187 treatment resulted in the release of labeled collagen in a dose-dependent manner that was rapid, detected as early as 2 hours, and reached a peak by 4–8 hours (data not shown). These observations indicate that LPS and calcium ionophore activate chondrocyte-dependent collagen release. The results of trypan blue dye exclusion studies ruled out the possibility of chondrocyte lysis as the cause of collagen release by activated chondrocytes. The rapid timing of the release of collagen suggests the involvement of chondrocyte-derived oxidative stress in collagen degradation.

Measurement of lipid peroxidation activity in OA chondrocytes.

We have previously shown that collagen degradation by activated articular chondrocytes in our degradation model assay is essentially mediated by chondrocyte lipid peroxidation (28). Lipid peroxidation activity was measured in 7 samples of primary OA chondrocytes treated with cis-parinaric acid (10 μM). Serial spectrofluorometric reading of resting and activated (by calcium ionophore [4-bromo-, a nonfluorescent species] or LPS) OA chondrocytes in the presence of physiologic concentrations of vitamin E showed lipid peroxidation activity in all samples tested. However, in some cell samples, lipid peroxidation activity was not enhanced by treatment with LPS or calcium ionophore, suggesting that in these 4 chondrocyte specimens, lipid peroxidation activity was already maximally elevated, whereas in the other 3 samples tested, OA chondrocytes showed a minimal response to the activating stimulus. Physiologic concentrations of vitamin E were shown to diminish the loss of fluorescence in both resting and activated OA chondrocytes (Figure 2). These results indicate that a measurable level of lipid peroxidation activity in human OA chondrocytes can be detected and that in more than 50% of the samples tested, the level was already maximally elevated.

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Figure 2. Measurement of lipid peroxidation in osteoarthritic (OA) chondrocytes over time. Primary OA chondrocytes were treated with cis-parinaric acid (10 μM) for 60 minutes. Parinaric acid was excited at 325 nm, and fluorescence signals were collected at 405 nm in 37°C water-jacketed cuvettes in the presence or absence of vitamin E (50 μM) at the indicated intervals. Results from a representative experiment are shown.

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Dichlorofluorescein oxidation in OA chondrocytes.

We monitored DCF oxidation activity in 8 OA chondrocyte specimens. A typical example is presented in Figure 3, where a progressive increase in the fluorescence of cells treated with DCF can be seen, an effect that was further enhanced by LPS treatment. Physiologic concentrations of vitamin E diminished the oxidation of both untreated control and LPS-activated OA chondrocytes. In contrast to the lipid peroxidation activity described above, all OA chondrocyte samples showed spontaneous and inducible DCF oxidation activity. These results suggest that human OA chondrocytes show intracellular oxidative activity that is enhanced by treatment with LPS. Furthermore, treatment with physiologic concentrations of the antioxidant vitamin E diminished the oxidative activity.

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Figure 3. Measurement of 2′,7′-dichlorofluorescein (DCF) oxidation by osteoarthritic (OA) chondrocytes in the presence and absence of lipopolysaccharide (LPS) and vitamin E. Detached primary OA chondrocytes were incubated at 37°C with 5 μM DCF diacetate. DCF oxidation in LPS-stimulated (200 μg/ml) chondrocytes in the presence and absence of vitamin E (Vit E; 50 μm) was monitored by fluorometry (excitation 488 nm; emission 525 nm). DCF oxidation was also measured in control chondrocytes in the presence of vitamin E. Results from a representative experiment are shown.

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Presence of malondialdehyde and hydroxynonenal adducts in OA cartilage.

Fourteen samples of OA cartilage obtained at the time of hip or knee joint replacement and 3 samples of normal cartilage obtained at the time of finger amputation (1 sample) or arthroscopic examination of the knee joints (2 samples) were subjected to immunohistochemical analysis. Monoclonal antibodies that detect MDA and HNE lysine adducts were used to detect the presence of aldehydic adducts in the tissues.

Treatment of cartilage sections in the absence of primary antibody showed no immunoreactivity (Figure 4). Cartilage sections treated with either MDA2 or NA59 antibodies showed immunospecific reactivity, suggesting that these monoclonal antibodies reacted to specific antigenic sites that were generated by in vivo lipid peroxidation activity in the OA tissue samples. Specific immunoreactivity of variable intensity was observed in all 14 OA tissue sections. Reactivity of MDA and HNE adducts was detected on the cartilage surface, immediately beneath the surface, and in the cartilage matrix in all 3 zones (superficial, middle, and deep). Marked staining of chondrocytes and pericellular matrix was observed in the sample from OA patient 4. Enhanced immunostaining was observed at intra- and intercellular matrix sites, around the cysts, and particularly at sites of OA lesions, such as erosion and fissures.

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Figure 4. Detection of malondialdehyde (MDA) and hydroxynonenal (HNE) lysine adducts in osteoarthritic (OA) and normal cartilage. OA and normal (old age) cartilage sections were enzyme treated and were then either left untreated (control) or were treated with MDA2 (1:200 dilution) or NA59 (1:20 dilution) monoclonal antibodies. Antibody binding was determined by immunohistochemical reaction as described in Materials and Methods. Control sections show only a very faint surface reactivity. Immunoreactivity to both MDA2 and NA59 is very strong, and the staining is immunospecific. The cartilage surface, chondrocytes, surface erosions, fissures, the pericellular matrix, the intra- and intercellular matrix, and the matrix around the cysts show positive staining, which suggests the presence of MDA and HNE lysine adducts. These findings indicate the formation of aldehyde protein adducts in OA cartilage and, therefore, suggest a role in OA pathogenesis. In contrast, normal cartilage shows only a very faint staining of the cartilage surface. Chondrocytes in normal cartilage show little immunoreactivity. H&E = hematoxylin and eosin. (Original magnification × 100.)

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In contrast to the specific immunostaining seen in the OA tissues, only faint immunostaining of the cartilage surface was observed in sections of normal cartilage. These observations suggest that OA cartilage shows the presence of in vivo markers of lipid peroxidation activity. Specifically, these markers were detected at the sites of OA lesions, in and around chondrocytes, and in the matrix, suggesting its pathogenic role.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In the present study, we developed a human OA chondrocyte-dependent model of collagen degradation, similar to one we previously described in which we used normal lapine chondrocytes (28). The human chondrocyte model showed exquisite sensitivity. The release of 3H-labeled material into the medium corresponded to the potential oxidative damaging effects of hydrogen peroxide. Of the various agonists tested to initiate a respiratory burst in OA chondrocytes, only calcium ionophore and LPS rapidly and in a dose-dependent manner induced the release of 3H-proline–labeled material into the medium. The release of labeled matrix was not a result of cell death, but demonstrated characteristics of a cell-dependent oxidative degradative process in the extracellular matrix, such as the rapid cause-and-effect relationship of matrix catabolism. Physiologic concentrations of vitamin E inhibited collagen degradation (data not shown). These observations confirm that this model can be a useful tool with which to study the mechanisms of collagen degradation in OA.

To demonstrate the role of chondrocyte lipid peroxidation, we directly measured lipid peroxidation activity in cis-parinaric acid–treated primary OA chondrocytes. These studies showed constitutive and inducible lipid peroxidation activity in OA chondrocytes. However, 4 of the 7 samples tested showed maximal levels of lipid peroxidation activity, which was not enhanced by treatment with LPS or calcium ionophore, whereas the remaining samples showed a minimal response to the activating agent. Thus, lipid peroxidation activity in OA chondrocytes was maximally elevated in more than 50% of the OA chondrocyte samples tested, to the extent that further stimulation with agonist was not possible.

Why do OA chondrocytes have such high levels of lipid peroxidation activity? It is possible that the enhanced lipid peroxidation activity in OA chondrocytes is the result of an altered lipid profile. Bonner et al (33) first documented that lipids, especially polyunsaturated fatty acids (PUFAs), accumulate with aging of normal cartilage. Adkisson et al (34) showed that normal cartilage expresses low levels of n-6 PUFAs and high levels of n-9 fatty acids. High levels of n-9 PUFAs found in young cartilage are progressively depleted with increasing age, and this depletion of n-9 PUFAs is accompanied by a steady increase in the levels of n-6 PUFAs. Similar changes are particularly pronounced in OA cartilage (34). In several models of degenerative arthritis, lipid accumulation generally precedes local tissue degeneration (35, 36). Silberberg and colleagues (37, 38) demonstrated an increase in the incidence of age-dependent OA in the C57 inbred strain of mice fed a diet high in saturated fatty acids. Dietary lipids also modify the fatty acid composition of cartilage and isolated chondrocytes (39).

The tissues from degenerated joints exhibit an acceleration of metabolism, an effect that can be reproduced in vitro with normal chondrocytes supplemented with exogenous essential fatty acids (40, 41). Lippiello et al (42) observed that the levels of fatty acids were markedly enhanced in OA cartilage in association with an increasing degree of OA lesions. Certain rare forms of osteochondrodysplasia associated with precocious degenerative joint disease are characterized by the accumulation of lipids in chondrocytes (43, 44). Taken together, the evidence suggests that the distribution and content of lipids in cartilage change during aging and OA. Lipids are essential for maintaining the integrity of the cell membrane. Lipid peroxidizability was shown to increase with the PUFA content in model membranes and cells (45). There is a strong body of evidence that supports a tissue-damaging role of lipid peroxidation (22).

Measurement of lipid peroxidation activity provides an assessment of only lipid-derived sources of oxidative stress resulting from the oxidation of lipids in cell membranes (22). To broaden this investigative approach, we used a DCF oxidation method to measure the overall oxidation activity in OA chondrocytes. DCF oxidation primarily measures the intracellular production of reactive oxygen intermediates, such as hydrogen peroxide, peroxidases, ROS other than hydrogen peroxide, and reactive nitrogen species (46–48). We observed DCF oxidative activity in all OA chondrocyte samples examined. In contrast to lipid peroxidation activity, all OA specimens showed spontaneous and inducible DCF oxidation activity. These observations indicate that lipid peroxidation activity may be selectively pushed to near-maximal levels in OA chondrocytes, whereas the overall oxidative activity in OA chondrocytes may show an activation-dependent response pattern. It is possible that the oxidative activity measured by DCF in OA chondrocytes may be abnormal, but we were unable to perform this measurement in normal articular chondrocytes for a comparison because of insufficient tissue.

Because MDA and HNE, which are specific and major toxic products of lipid peroxidation, are responsible for tissue damage, their identification in tissues provides specific molecular markers of in vivo tissue damage mediated by lipid free radicals (22–24). Aldehydic products of lipid peroxidation liberated outside the cells oxidizes the extracellular matrix components, resulting in fragmentation, modification, aggregation, and protein conformation changes and eventually leads to alterations in tissue functioning. Using immunohistochemical methods, we identified MDA and HNE protein adducts in OA tissue sections. The binding of monoclonal reagents to tissue was immunospecific, indicating that these aldehydic adducts were indeed present in OA tissue and were derived in vivo. Furthermore, aldehydic adduct activity was detected at the sites of OA lesions, such as around cysts and erosions, and at fissure sites. Aldehydic adduct immunoreactivity was also present at intra- and intercellular matrix sites, indicating evidence of lipoxidation damage in matrix tissue. In addition, the immunoreactivity was present around individual chondrocytes and chondrocyte clusters, suggesting that chondrocytes may be the source of lipid peroxidation in cartilage. In contrast, normal cartilage sections showed nonspecific reactivity only on the cartilage surface. Normal cartilage chondrocytes showed little immunoreactivity. Taken together, these observations indicate that the MDA and HNE adducts were distributed in a lesion-specific pattern in OA cartilage, suggesting their pathogenic role.

Using specific immunoreagents that detect intact, MMP-specific cleavage or denatured collagen, Hollander et al (49) observed a progressive gradient of cartilage damage from the top to the deep zone. In the present study, no such gradient for aldehydic adduct immunoreactivity was observed. The aldehydic adduct reactivity was detected in all 3 zones (superficial, middle, and deep). The progressive gradient of damage from the top to the deep zone of cartilage was interpreted as indicating that the pathology of OA is initiated at the top of the cartilage and gradually progresses to the deep zone (49). Our observations suggest that lipoxidation-induced damage is initiated throughout the entire cartilage thickness.

The unique distribution of lipids in cartilage, which changes significantly with age, the influence of the dietary intake of lipids, and the consequent lipid peroxidizability suggest a role of lipoxidation in the pathology of OA. The presence of aldehydic adduct markers, which are specific for lipid peroxidation, in OA cartilage, as observed in the present study, provides direct molecular evidence that lipoxidation plays a role in the pathogenesis of OA. Enhanced levels of lipid peroxidation activity in OA chondrocytes also indicate a role of lipid peroxidation in OA. The chondrocyte-dependent oxidative damage to cartilage collagen shown in our in vitro degradation model also supports a role of lipoxidation. In the Framingham Knee Osteoarthritis Cohort study (50), the population with a medium to higher intake of vitamin C, β-carotene, and vitamin E had a reduced risk of progression of knee OA, as assessed radiographically. Antioxidants have not been rigorously tested in the prevention or treatment of OA. The results of our studies (refs. 27–29 and the present study) as well as those of other studies on the role of nitric oxide (21) and redox status (14) in chondrocyte aging indicate that antioxidant therapies may prove useful in the prevention and/or treatment of cartilage aging and OA.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES