Cyclooxygenase inhibition lowers prostaglandin E₂ release from articular cartilage and reduces apoptosis but not proteoglycan degradation following an impact load in vitro

Articular cartilage is a highly specialised connective tissue that covers the ends of long bones in diarthrodial joints. The tissue protects the joint by distributing applied loads and providing a low-friction, wear-resistant, lubricated surface to facilitate movement. The cartilage matrix consists of collagen fibres that reinforce a proteoglycan gel. The main protoeoglycan is aggrecan, which comprises a protein core highly substituted with polysulfated glycosaminoglycan (GAG) side chains.

Traumatic joint damage, such as may be sustained in a road traffic accident or a sporting injury, is a known risk factor for the subsequent development of secondary osteoarthritis (OA) [1]. Injury can result in progressive cartilage loss causing pain, swelling, inflammation and joint immobility. Ultimately, a joint replacement may be required. However the processes resulting in cartilage breakdown following injury and the ability of the tissue to repair itself are poorly understood. In humans, studies have shown elevated levels of breakdown products from cartilage matrix many years after injury [2‐4]. The relationship between joint injury and OA development has also been demonstrated in various animal models both in vivo and in vitro [5, 6].

Under normal physiological loading, articular cartilage is subjected to a variety of stresses. These biomechanical factors are believed to stimulate chondrocyte metabolism, providing a mechanism for the cartilage to adapt to the demands imposed by the body. However, in abnormal or injurious joint loading the balance between cartilage matrix synthesis and degradation is disturbed [7], resulting in tissue breakdown and the risk of progression of OA.

There is considerable evidence that the cytokine interleukin-1 (IL-1) plays an important role in OA, being up-regulated in OA synovium and cartilage [8, 9]. IL-1β expression in articular cartilage is also regulated by mechanical factors [10]. It induces a catabolic cascade involving the cyclooxygenase (COX) enzymes; two isoforms of which, COX-1 and COX-2, catalyse the conversion of arachidonic acid to prostaglandins (PG), the major pro-inflammatory product being prostaglandin E₂ (PGE₂) [11]. COX-1 is the constitutive form of the enzyme, naturally expressed at low levels and essential to the normal function of many tissues, whereas COX-2 is the inducible form, which is commonly up-regulated following an insult to the tissue [12]. Consequently, PGE₂ has been found to be elevated in cartilage, synovium and synovial fluid in OA joints [13, 14] and also in normal cartilage by prolonged static mechanical loads [15]. Similarly COX-2, but not COX-1, has been shown to be up-regulated in chondrocytes of OA cartilage [16]. This COX-2/PGE₂ pathway is of major interest in OA as the first line of treatment in this disease is the use of the non-steroidal anti-inflammatory drugs (NSAIDs) for pain relief. These drugs inhibit the activity of COX [17]. The non-selective NSAIDs inhibit both COX-1 and COX-2 (e.g. indomethacin) and more recently NSAIDs have been developed that are more selective for COX-2 (e.g. celecoxib) exhibiting fewer unwanted side effects.

Several studies, both in animal models [18] and in human joints [19, 20], have shown that apoptosis (programmed cell death) is an important factor in the progression of OA. A positive correlation exists between severity of OA and percentage of apoptotic cells [21]. Apoptosis occurs following mechanical injury [22‐26] and is thought to be initiated by IL-1β (in the presence of tumour necrosis factor (TNF) α; [27]), which in turn activates the caspase cascade [28]. As IL-1β is also involved in activating the COX/PGE₂ pathway and PGE₂ is reported to induce apoptosis in bovine articular chondrocytes [29], the prostanoid may have a role in the increased apoptosis observed following trauma.

In an attempt to understand the physical and biochemical changes occurring in articular cartilage following trauma, an in vitro impact model has been developed [7, 30]. This consists of an instrumented drop tower, which enables an explant of cartilage to be subjected to a controlled impact load [31].

The aim of this study was to ascertain whether PGE₂ was released by articular cartilage chondrocytes following an impact load, and whether COX-2 inhibition using NSAIDs could provide a chondroprotective role by preventing matrix degradation. In addition, the role of COX inhibitors on trauma-induced chondrocyte apoptosis was investigated.

PGE₂ levels were increased 22-fold in the medium of explants at 3 days (p = 0.012) following impact compared to unloaded controls. By 6 days this increased further to 27-fold (p = 0.004) (Figure 1a). In the presence of celecoxib and indomethacin, the PGE₂ levels were reduced in a dose-related manner both at 3 days and, more significantly, 6 days (Figure 1b). At the highest concentration (10 μM) the levels were reduced to those of the unloaded controls by both inhibitors at both timepoints. The baseline concentrations in the medium of control human cartilage explants were similar to those in other in vitro studies [37‐39].

The concentration of GAGs in the medium was significantly higher in the loaded explants than in the unloaded controls at both day 3 (p = 0.010) and day 6 (p = 0.003) following impact (Figure 2a). However the addition of celecoxib or indomethacin to the culture medium had no effect on the release of GAGs from the cartilage matrix at either 3 days or 6 days (Figure 2b). Therefore, in this study, COX inhibition following impact load did not prevent proteoglycan depletion of cartilage.

An impact load increased the percentage of apoptotic cells compared with unloaded controls (p = 0.022). The apoptotic chondrocytes were evenly distributed throughout the zones of the loaded cartilage sections (Figure 3). Addition of the COX inhibitors to the medium reduced the percentage of impact-induced apoptosis at all doses and reached significance at 0.1 μM celecoxib (p = 0.034) and 10 μM indomethacin (p = 0.032) (Figure 4).

A single impact load resulted in a marked increase in PGE₂ release, the number of apoptotic cells, and the concentration of GAGs in the culture medium. Could the elevated levels of PGE₂ lead directly to these other changes and hence provide a target for early therapeutic intervention to delay or, even, prevent later secondary OA?

Tissue damage resulted in an increase in PGE₂ released to the culture medium so that by day 3 after impact, the concentration was more than 20 times higher than in control groups. The detailed course over time of this was not studied, but release continued – albeit at a slower rate – over the next 3-day period. This release could be inhibited in a dose-dependent fashion by both celecoxib and indomethacin. Apoptosis was also reduced but not in the same pattern. Indomethacin halved the number of apoptotic cells at a concentration of 1 μM and halved it again at 10 μM. In contrast, celecoxib approximately halved the number of apoptoptic cells but concentration appeared to have little effect; maximal effect was found at 0.1 μM, but this was not significantly different from any of the other concentrations used. Despite the reduction in apoptosis, neither inhibitor had any effect on matrix degradation, as indicted by there being no change in GAG release.

The release of PGE₂ by articular chondrocytes and its inhibition by indomethacin is well established [40]. Once released, however, the effects of this prostanoid on the cartilage matrix are rather unclear. PGE₂ is reported to have anabolic effects on cartilage: increasing proteoglycan and collagen synthesis [41], stimulating proliferation and aggrecan synthesis [42], up-regulating glucocorticoid receptors [43] and, at low concentrations, stimulating collagen II synthesis. All these effects are possibly mediated by insulin-like growth factor 1 (IGF-1) via an autocrine loop [44]. However, this response was reported to be biphasic in chondrocytes in vitro because at higher concentrations of PGE₂ collagen synthesis was reduced precipitously [44]. In earlier studies we showed that a static load of 1 MPa on human articular cartilage explants in vitro resulted in an increased expression of IL-1β [45] and PGE₂ [15], though cyclic loading produced no measurable change in either IL-1β or PGE₂ suggesting that this is a pathological response. In this study, we show that physical injury to cartilage following impact results in a significant increase in PGE₂.

The increase in the percentage of apoptotic cells was similar to that we measured previously with human cartilage using the same drop-tower model [46]. The role of PGE₂ in promoting apoptosis, however, remains unclear. Addition of exogenous PGE₂ to bovine articular chondrocytes in vitro has been shown to cause apoptosis through a cAMP-dependent pathway [29]. However in human chondrocytes from OA cartilage, Notoya et al. [47] found that PGE₂ had no effect on chondrocyte apoptosis itself, but the prostanoid enhanced apoptosis induced by exogenous nitric oxide and this effect could be prevented by COX-2 inhibition. In contrast, PGE₂ has been reported to protect chondrocytes from apoptosis induced by actinomycin-D [48]. In addition, PGE₂ is a chondrocyte growth inhibitor that requires NO for its production [49], and both PGE₂ and NO are downstream mediators of IL-1. The partial, dose-independent reduction we found suggests that a cofactor role for PGE₂ is possibly more likely than a direct effect. Further studies are required to investigate the role of NO and IL-1β.

Hashimoto et al. [21] linked chondrocyte apoptosis to matrix destruction. In this study however, the inhibition of apoptosis by the addition of celecoxib or indomethacin was not found to reduce the amount of GAGs, the breakdown products of cartilage proteoglycans, lost from loaded explants. This result is similar to that we previously found, culturing human articular cartilage explants with a broad spectrum caspase inhibitor (Z-VAD-FMK) reduced the percentage of impact-induced apoptotic chondrocytes but was unable to reduce the amount of GAGs released into the medium following impact [50]. Since articular cartilage is not vascularised and does not contain mononuclear phagocytes, there is no apparent mechanism for removal of apoptotic bodies following chondrocyte apoptosis. It has been shown that these apoptotic bodies express properties that contribute to pathologic matrix destruction [27]. Therefore, although matrix degradation, as measured by GAG release, was unchanged in this study, COX inhibition may still have chondroprotective effects by reducing the percentage of impact-induced apoptotic cells remaining in the tissue (and, therefore, there would be fewer potentially destructive apoptotic bodies). Together, though, these studies indicate that apoptosis alone is not driving matrix breakdown and that, once started, degradative enzymic activity may not be under direct cellular control. This may be because these enzymes are sequestered extracellularly in inactive forms and activation leads to a positive feedback pathway that is then out of direct control of the cells. Alternatively, enzymic activation is controlled by a different signalling pathway, though this then raises the question as to why the remaining cells cannot recognise this activity and inhibit it? The cells have been shown to be able to increase their levels of matrix biosynthesis following impact-induced damage [7] so perhaps matrix breakdown is part of a repair response to try to remove damaged tissue and replace it. Assuming, however, that it is important clinically to reduce matrix degradation, a two-pronged approach to treating damaged tissue would then be required; one agent to rescue the cells from apoptosis and another to inhibit enzymic degradation. The complexity of these mechanisms requires further investigation, in particular addition of exogenous PGE₂ and subsequent measurement of proteoglycan synthesis in this system would demonstrate any anabolic effects. However, we have shown that inhibiting PGE₂ in impact-damaged cartilage at least partially prevented the increase in apoptotic cells otherwise found after 6 days.

Both celecoxib and indomethacin could abolish the increase in PGE₂. Celecoxib is 375 times more selective for COX-2 than COX-1 [51] whereas indomethacin is generally considered to be non-selective. Several studies have shown that COX inhibitors have an effect on cartilage metabolism. COX-2 inhibition has no direct effect on normal, healthy cartilage, but in the presence of IL-1β or TNFα it restores proteoglycan turnover [52]. Additionally, Hajjaji et al. [53] found that celecoxib had a favourable effect on the metabolism of proteoglycans and hyaluronic acid in samples of OA cartilage in vitro. Non-selective NSAIDs have differing effects on cartilage metabolism. Some stimulate, some have no effect, and others – including indomethacin – inhibit matrix synthesis [54, 55]. Mastbergen et al. [38] have shown that in OA cartilage, NSAIDs with low COX-2/COX-1 selectivity exhibit adverse effects whereas high COX-2/COX-1 selective NSAIDs either had no effect or had reparative properties. Since in our study the non-selective NSAID indomethacin inhibited impact-induced apoptosis, future experiments with an experimental selective COX-1 inhibitor (i.e. SC-560) may be of use to investigate the precise role of COX-1 in this impact model. In patients, selective COX-2 inhibition would appear to confer beneficial effects on articular cartilage metabolism while avoiding the harmful effects of COX-1 inhibition, such as gastric irritation and inhibition of matrix synthesis, though possible cardiovascular effects of COX-2 inhibition have yet to be resolved.

This study has shown that an impact load on articular cartilage results in a marked increase in PGE₂ synthesis. This increase could be abolished by both the selective COX-2 inhibitor, celecoxib, and by non-selective indomethacin. Chondrocyte apoptosis, induced by impact, was also reduced by COX-2 inhibition. No change was observed in the release of GAGs from the explants in the presence of these inhibitors, however, suggesting that the COX/PGE₂ pathway is not directly responsible for cartilage breakdown following traumatic injury. The inhibition by COX inhibitors of PGE₂ release following trauma may provide an opportunity for early clinical intervention to reduce cell death from apoptosis. Our in vitro study suggests that it is unlikely, however, that COX-2 inhibition alone would slow down or prevent the development of secondary osteoarthritis.