The proteoglycan 4 (PRG4) gene (1) encodes for mucin-like O-linked glycosylated proteins, including lubricin (2) and superficial zone protein (3). PRG4 proteins, collectively referred to as PRG4, are synthesized and secreted by cells within articular joints, including superficial zone articular chondrocytes (3) and synoviocytes (4). PRG4 is present in synovial fluid (SF) (5) and at the articular cartilage surface (6). PRG4 acts as a boundary lubricant; it mediates friction during cartilage-on-cartilage contact between the articular surfaces, where lubrication is provided by molecular interactions at the surface (7). While PRG4 alone is an effective boundary lubricant, it also acts synergistically with hyaluronan (HA) to further reduce friction to levels approaching that of whole SF (8). HA, a linear polymer of repeating disaccharides composed of D-glucuronic acid and D-N-acetylglucosamine (9), is another boundary lubricant that is present in SF (8). It appears that both PRG4 and HA are critical to the boundary-lubricating function of human SF.
Changes in the PRG4 composition of human SF after acute injury and in osteoarthritis (OA) have been observed. Average concentrations of PRG4 in normal SF between 35 and 250 μg/ml (10–15) have been reported. PRG4 concentrations have been observed to decrease significantly after anterior cruciate ligament injury, returning to normal within ∼1 year (12). Concentrations have been observed to increase after intraarticular fracture (11), remain normal after internal derangement (13), and be elevated in late-stage OA (10, 14). However, animal models have suggested that the PRG4 concentration in SF and its presence in the superficial zone can decrease in secondary OA (16–18). Along with an altered lubricant composition, compromised boundary-lubricating ability was observed after intraarticular fracture (11). However, no difference between the steady-state boundary-lubricating ability of OA and normal SF has been observed (14, 19). Mutations in the PRG4 gene in humans cause an autosomal-recessive disorder known as camptodactyly-arthropathy–coxa vara–pericarditis (CACP) syndrome (20). SF from these patients is void of PRG4 and fails to lubricate (21). Collectively, these findings in normal, injured, and diseased human SF suggest that SF deficient in PRG4 lacks normal boundary-lubricating ability.
The HA composition of human SF has also been observed to change with injury and disease. Average normal concentrations of HA in human SF samples range between 1.8 and 3.33 mg/ml (11, 13, 14, 19, 21, 22). The HA concentration in human SF has been observed to remain normal in internal derangement injuries (13), to significantly decrease with intraarticular fracture (11), effusive joint injury, and arthritic disease (22–24), and to remain normal during OA (14, 19, 25) and CACP syndrome (21). The HA concentration has also been observed to be correlated with the age of the patient (25). The molecular weight distribution (MWD) of HA has been shown to range continuously between 27 kd and 10 Md in normal SF, peaking between 6 and 7 Md (25–28). The MWD of HA has been observed to shift to the lower range during injury (13) and OA (14), but has also been observed to remain constant between normal SF and OA SF (25). The HA MWD in SF is of interest for the potential difference in lubricating ability and interaction with PRG4 of different MW species of HA (29). It has been observed that HA supplementation of HA-deficient equine SF after acute injury was able to restore compromised boundary-lubricating ability (30).
Intraarticular injection of HA is currently used to treat OA. Commercially available formulations of intraarticular HA range from 0.5 to 6 Md and from 8 to 15 mg/ml (31, 32). It has been demonstrated in injury models of OA in rats that intraarticular injection of PRG4 protects against cartilage degeneration (33–35). The potential application of PRG4 as a new and improved therapy for postinjury and OA knee joints, as well as for maintenance of healthy joints, is promising. However, it is unclear if PRG4 concentrations remain normal in OA SF, and the biomechanical effects of supplemental PRG4 on the boundary-lubricating ability of SF, especially SF deficient in PRG4, in normal human cartilage are unknown.
The objectives of this study were therefore to quantify the PRG4 and HA content in SF samples from normal donors and patients with chronic OA and to assess the human cartilage boundary–lubricating ability of PRG4-deficient OA SF as compared to that of normal SF, with and without supplementation with PRG4 and/or HA.
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The findings of this study provide insight into the molecular basis for altered cartilage boundary–lubricating ability of OA SF. These results are consistent with the notion that PRG4 concentrations can vary considerably among OA patients as well as among normal donors. Furthermore, they indicate that normal PRG4 levels may not be present in all SF from patients with chronic OA and suggest that there is a subpopulation of OA patients whose SF is deficient in PRG4, associated with diminished cartilage boundary–lubricating ability. These results further emphasize that PRG4 is a critical boundary lubricant and is required for normal joint lubrication.
The ELISA used to measure PRG4 levels extends previous PRG4 quantification methods. In this assay, human SF was treated with Sialidase A-66 prior to quantification. HRP–PNA has previously been used as a capture reagent in an SF sandwich ELISA (10, 12), without neuraminidase digestion. Due to ∼46% capping of human PRG4 glycosylations with sialic acid (41), the PRG4 concentration measured with and without neuraminidase digestion may differ. Digestion of SF and control PRG4 with Sialidase prior to ELISA measurement increased the signal strength in both. The PRG4 concentration in samples not treated with Sialidase could not be accurately determined from similarly treated controls due to the very low signal obtained, as the assay is optimized for controls and samples treated with Sialidase. Potential HA–PRG4 interactions that may interfere with antibody recognition of PRG4 were disrupted using hyaluronidase, as previously performed in a quantitative Western blot method (11, 13, 14). Several antibodies have been used with previous PRG4 quantification methods (11, 13, 42). This ELISA recognizes high MW PRG4 species (>345 kd, including multimers, identified by LPN capture ) with glycosylations (identified by HRP–PNA detection) (37), both of which are important for functionality (41). Finally, SF samples were stored with PIs before quantification; sample storage without PIs may result in an underestimate if PRG4 has degraded during storage. Addition of PIs had no effect on the PRG4 signal as measured by ELISA (data not shown).
The PRG4 concentrations obtained for normal SF in this study are consistent with those measured in previous studies. Furthermore, the range of PRG4 concentrations measured in normal SF (129–450 μg/ml) reflects the previously reported wide range of PRG4 concentrations in normal SF (10–15). Large variability of these values in SF from patients with joint disease has been reported (276–762 μg/ml) (13–15) and was also observed in the present study (range in all OA SF samples examined 95–426 μg/ml). It should be noted that none of the OA donors with PRG4-deficient SF had a history of recent injury, which is known to affect the PRG4 concentration (12). The PRG4 concentration has previously been observed to increase with OA (10, 14, 15), and several samples with normal to elevated concentrations of PRG4 were also identified in this study (data not shown).
While a decrease in PRG4 levels with OA has not previously been reported in humans, a decrease in SF PRG4 levels with secondary OA has been observed in guinea pigs (16, 17), as has a decreased presence of PRG4-positive chondrocytes in the superficial zone after meniscectomy in an ovine model (18). A decrease in lubricating ability of SF from patients with rheumatoid arthritis (RA) has been observed (19), as has a classification of RA patients based on high and low levels of PRG4 expression in the synovium (43). Possible mechanisms for decreased PRG4 concentrations in the PRG4-deficient OA SF samples identified in this study include decreased expression/synthesis of PRG4, increased degradation of PRG4 (12), or increased loss of PRG4 from the joint capsule through an inflamed synovium (44, 45). Further investigation into the characteristics of the study patients would contribute to the understanding of the mechanism underlying PRG4 deficiency. Increased friction due to PRG4 deficiency is a clinically relevant issue, as friction and wear are thought to be coupled at the articular surface (21).
The normal HA concentration and shift to lower MW HA observed in the PRG4-deficient OA SF samples is consistent with the findings of previous studies (13, 14, 19, 25). The HA concentrations measured are lower than those observed in previous studies of human SF. Concentrations ranged from 0.11 to 0.96 mg/ml (normal) and from 0.23 to 2.69 mg/ml (OA, not friction tested) in the present study, and from 1.8 to 3.33 mg/ml (normal) (11, 13, 14, 19, 21, 22) and from 0.1 to 1.3 mg/ml (diseased) (22, 24) in the literature. There was no statistically significant difference in the HA concentration between OA SF and normal SF, as previously reported (14, 19, 25). HA concentrations measured for bovine SF (range 0.32–0.79 mg/ml) (data not shown) are consistent with previously measured values (∼0.5 mg/ml) (46).
Both PRG4 deficiency and a shift toward a lower MW of HA in some SF samples from patients with chronic OA were observed in the current study. Previous studies have demonstrated that the boundary-lubricating ability of HA alone increases with increasing MW (30); however, the synergistic boundary-lubricating ability of HA with PRG4 is not dependent on MW (29). These studies together suggest that treatment with PRG4 could negate the deleterious effects of a shift toward HA of low MW in OA SF and prevent alterations in boundary-lubricating ability (29). Completing the biochemical and biomechanical characterization on human SF samples with normal and elevated PRG4 concentrations (identified but not described) will help to clarify this relationship.
In this study, a statistically significant effect of additional supplementation with HA on the boundary-lubricating ability of PRG4-deficient OA SF was not observed. However, as PRG4 supplementation of PRG4-deficient samples was of interest and was performed first, the effect of HA supplementation alone in human SF remains to be fully elucidated. Other studies have shown that HA supplementation of acute-injury equine SF deficient in HA restored compromised boundary-lubricating ability (30). Alterations in the boundary-lubricating ability of human SF are of great interest, as small increases in friction have been observed to be associated with increased wear at the articular surfaces (21).
This study is unique in that both normal cartilage and normal SF were obtained for use as controls. Normal cartilage was obtained from macroscopically normal areas of femurs from donors who had not been taking antiinflammatory drugs. The coefficients of friction for boundary lubrication obtained for normal SF on normal cartilage (<μkinetic, Neq> = 0.025) are consistent with the coefficients of friction measured for bovine SF on bovine cartilage in an identical test (<μkinetic, Neq> = 0.025) (8); this supports the use of normal cartilage. Furthermore, total protein concentrations measured in normal SF were consistent with previously reported values (range 18–28 mg/ml) (44, 45) and were lower than those measured in OA SF. The volumes of normal SF obtained in the present study were generally within the reported range of normal (0.5–4 ml) (45). The OA SF volumes were significantly higher, as expected. It should be noted that in this study, no correlation between the volume of SF aspirated and the PRG4 concentration was observed.
Previous studies using this in vitro cartilage-on-cartilage friction test confirmed that up to 5 sequential tests could be conducted on a single osteochondral pair over 5 days, with overnight storage at 4°C between tests, without degradation of the samples. To account for any potential carryover effect of test lubricants and to isolate the effect of PRG4 supplementation, the test sequence we used was chosen according to the order of presumed increasing lubricity. The HA and PRG4 used in this study were representative of those in native human SF and have been used in other studies (29). The concentration for PRG4 supplementation was selected based on values previously observed to provide boundary lubrication (8), values previously reported in human SF (10–15), and preliminary measurements in normal SF by ELISA (as additional normal SF samples are obtained and characterized on an ongoing basis). The HA concentration for supplementation was selected based on preliminary measurements in normal SF by ELISA, and a MW of 1.5 Md was selected as it is in the range of commercially available formulations of HA for intraarticular injection (31, 32). Furthermore, 1.5-Md HA has previously been shown to provide boundary lubrication (29).
These findings support and significantly extend the observation that human SF deficient in PRG4 demonstrates decreased boundary-lubricating ability. The PRG4-deficient OA SF samples identified had normal HA concentration, altered HA MWD, and decreased lubricating ability. This suggests that the MWD of HA may be important and that low MW HA alone is not sufficient to provide normal boundary lubrication. Moreover, it provides further motivation to study PRG4–HA interactions in SF. PRG4 has been observed to exist in both a disulfide-bonded multimeric form and a monomeric form, which may affect its lubricating function (36). Future studies determining the multimer-to-monomer composition of PRG4 in normal SF and its alterations with OA will provide further insight into this fundamental joint lubrication mechanism.
Altered glycosylation patterns in OA, as observed between RA and OA, could be another source of variation in boundary-lubricating ability (37). The observations of this study are supported by in vivo studies by other research groups demonstrating that intraarticular injection of PRG4 into a rat model of injury-induced OA can prevent cartilage degeneration (33, 34). These results taken together with those of the present study suggest that in addition to postinjury patients, some chronic OA patients who have PRG4-deficient SF may benefit from PRG4 supplementation as a biotherapeutic agent to restore lubrication and maintain healthy joints.