Matrix metalloproteinase protein expression profiles cannot distinguish between normal and early osteoarthritic synovial fluid

There are likely several clinically recognized arthritic phenotypes, however, broadly speaking there are two main ‘types’ of arthritis: RA, a chronic inflammatory disease characterized by joint swelling, joint tenderness, and destruction of synovial joints [1] and OA, a heterogeneous group of conditions that lead to joint symptoms and signs which are associated with the defective integrity of articular cartilage [2]. Given the presence of auto-antibodies to a number of proteins such as citrullinated peptides (CP) and immunoglobulin (i.e. rheumatoid factor: RF) [1], RA has been appropriately classified as an autoimmune disease. There are a number of validated diagnostic tools and criteria that can predict the onset of RA, in some cases years before overt clinical manifestations. However, in the case of OA, no known (validated) early diagnostic test exists that can either predict the onset or eventual severity of the disease. To this end, a number of studies have used structural fragments of cartilage proteins as potential biomarkers of OA. Procollagen II C-propeptide upregulation, a precursor of COL II that appears as a repair response to damaged cartilage, has been localized in the articular cartilage of patients diagnosed with early OA [3]. Studies have shown this up-regulation is detectable in patient synovial fluid and serum, thus highlighting it as a potential biomarker for early OA [3]. Conversely, proteolytic COL II breakdown is key in the cartilage erosion witnessed in arthritic diseases. Fragments of COL II or COL II epitopes have also been highlighted as potential OA biomarkers, generally falling into one of three potential categories based on their origin from the native COL II molecule: cleavage neoepitopes, denaturation epitopes, or epitopes from the mature end of the molecule [4]. To date, however, no COL II/ProCOL II early OA diagnostic assays have entered the market due to the variability of COL II epitopes within normal and arthritic patient populations. Other historical biomarkers such as cartilage oligomeric protein (COMP) and hyaluronan (HA) [5, 6] have also been identified as early biomarkers, but have not been clinically adopted. More recently, profiling of the inflammatory response in OA patients is being investigated, however, this area of study is still in need of further investigation [7, 8]. Clinically speaking, only when evidence of joint space narrowing, cartilage loss, or osteophyte formation is obtained can the disease be clinically diagnosed in its later stages [2]. In this capacity, matrix metalloproteinases (MMPs) have been studied as potential indicators of the onset of arthritis as MMPs have been implicated in the loss of articular cartilage which occurs both in OA and RA [9, 10].

The chondrocyte extracellular matrix (ECM) of healthy and arthritic joints is constantly subjected to remodelling processes [11, 12]. This delicate balance between anabolism and catabolism of ECM components is mediated by interactions with synthetic and degradative molecules present in the joint environment (synovial fluid). In arthritic conditions, it is hypothesised that for some reason the catabolic potential within the joint environment overrides the anabolic, resulting in irreversible destruction within the joint. Many proteolytic enzymes have the ability to degrade the articular cartilage collagen scaffold including several of the MMPs [13, 14], as well as non-MMPs. MMPs are a family of tissue remodeling enzymes which, when activated, recognize specific sequences in ECM proteins and then cleave these proteins. MMP-1 is an interstitial collagenase (collagenase 1) produced mainly (but not exclusively) by synovial cells within the joint. MMP-1 efficiently degrades collagen types I and III during fibrolytic tissue remodeling, while also maintaining the ability to degrade type II collagen. MMP-2 is a gelatinase (gelatinase A) that is secreted by stromal cells beneath the synovial lining [15]. This MMP has the ability to degrade aggrecan, and extracellular matrix proteoglycan, as well as collagen types I, II, and III using a similar mechanism to MMPs of the collagenase subfamily. MMP-3 is a stromelysin (stromelysin 1) with a specificity to facilitate degradation of extracellular matrix proteoglycans such as versican and aggrecan. MMP-7, matrilysin, has been show to cleave proteoglycans and collagen III/IV/V/IX/X/XI when present in its active form [16]. MMP 7 in its active form has also been shown to activate pro-MMPs 1, 2, and 7 [16]. MMP-8, neutrophil collagenase, is secreted by neutrophils and MMP-9, gelatinase-B, is also secreted by neutrophils in addition to macrophages and synovial cells [15]. MMP-12, a metalloelastase secreted by macrophages, has the ability to breakdown collagen type IV, and proteoglycan, as well as various other ECM components [17]. MMP-13 is a collagenase (collagenase 3) produced by chondrocytes and has specificity for type II collagen, while to a lesser extent also degrades collagen types I and III as well as aggrecan. MMPs are inhibited by specific endogenous tissue inhibitors of metalloproteinases (TIMPs), which consist of four protease inhibitors: TIMP1, TIMP2, TIMP3 and TIMP4. Each TIMP binds with differential rates of interaction and affinity to a MMP, (1:1, 2:2 stoichiometry). TIMP-1 inhibits MMP-1, MMP-3 and MMP-9 [18–21]. TIMP-2 inhibits proMMP-2 [18, 22], however, TIMP-2 also has a bi-functional effect on MMP-2 since proMMP-2 activation requires low levels of TIMP-2 for activation, whereas a greater concentration of TIMP-2 inhibits MMP-2 [23]. TIMP-3 inhibits MMP-2 and MMP-9 [24], whereas TIMP-4 is a good inhibitor for all classes of MMPs without remarkable preference for specific MMPs [25].

In this study, the concentrations of specific MMPs (1, 2, 3, 7, 8, 9, 12, and 13) were quantified in the synovial fluid collected from normal joints and joints from patients clinically diagnosed with OA (early and advanced) or RA. Through PCA and K – means clustering analysis, reproducible and unique MMP expressions profiles were identified for normal, OA (advanced) and RA synovial fluid, however, normal and early OA synovial fluid cannot be distinguished from each other using this methodology. Utilizing high-through-put multiplexing methodologies with multi-variant statistical analysis allows for the visualization of changes within a certain marker between/within groups, but also provides the opportunity to observe if the interaction of many markers can better describe a phenotype of arthritic disease. This is of significance as distinct MMP profiles of disease may be exploited for alternative diagnostics as well as tracking disease activity/severity with therapeutic intervention.

Compared to MMP research in synovial fluid, few papers have examined and quantified TIMP protein expression in arthritic knee joints and compared this to TIMP expression in normal synovial fluid. Within the present study, no significant differences in TIMP expression (1, 2, 3, or 4) were detected between normal and early OA synovial fluid, which is consistent with the findings that normal and early OA synovial fluid could not be segregated based on MMP expression levels alone. Overall the published data seems to suggest that TIMP levels do not increase significantly with the severity of OA [34, 35], however, TIMP 1 levels decrease within injured joints [36]. Based on results present here and previous studies it would appear that the regulation of MMPs by TIMPs may be important in the progression of disease, however, it also appears that it cannot be clearly understood solely through the quantification and/or ratio of MMPs to TIMPs.

Using the Luminex platform, MMP-1, 2, 3, 7, 8, 9, 12, 13 and TIMP-1, 2, 3, 4 synovial fluid protein levels were quantified in normal, early OA, advanced OA and RA joints. Although there may be some concern over using cadaveric donations in this case, it was necessary to completely define the joints as ‘normal’. In some studies, patients requiring arthroscopic examination that do not demonstrate obvious pathology are considered normal, while in others synovial fluid is simply removed from joints of individuals that have no outward symptoms of arthritis.

For the present study, synovial fluid was chosen as MMP expression within the joint may or may not be correlated to that of serum/plasma [37–39]. Since cartilage degeneration of the knee takes place within the synovial boundary it makes sense to sample and test the synovial fluid for up/down-regulation of MMPs in the joint environment. Although cell sources of MMPs within the joint vary, for example synoviocytes (MMP 1, 2), chondrocytes (MMP 3, 7, 13), neutrophils (MMP 8, 9) and macrophages (MMP 12), with significant redundancy in expression of each MMP by multiple cell types, the results presented in this study (on MMP expression) correspond with previous cellular studies. Mature macrophages expressing MMP 12 are equally represented in the synovial membrane and fluid of OA and RA patients [40, 41], and in both disease states are significantly increased in number compared to normal synovial tissue [41], importantly though, MMP expression levels within a given cell type can also change with disease state [17]. Recently, through FACS analysis of synovial fluid of OA and RA patients it was identified that the levels of neutrophils are actually not significantly different [42]. Furthermore, past studies have demonstrated that neutrophils in RA presented diminished phagocytic activity compared to other types of arthritis [43]. Overall these results corroborate our MMP expression profiles (specifically MMP 8, 9 and 13) and suggest that although there are similar numbers of neutrophils in OA and RA, they may be expressing less MMPs in RA synovial fluid. In specific regards to MMP 2, a number of studies have clearly demonstrated that more fibroblasts are present in OA synovial fluid than RA in part validating this result [41, 44].

Although using MMP profiles within synovial fluid cannot distinguish normal vs. early OA knee joints, this methodology maybe applicable to drug/treatment testing, since this approach is based on a number of markers, even if one of two are differentially regulated within an individual, the overall profile of disease will not be significantly affected depending on the number of targets used. A number of MMP inhibitors have been developed to treat arthritic diseases with little or no effect, however, using this approach the overall effect of treatment (including related and ‘off’ targets) per individual profile could be determined using this high throughput approach. This would allow researchers, physicians and pharmaceutical companies to not only examine the effect of a specific drug on a specific target, but also visualize the effects more globally among a family of targets, which could lead to more efficient treatments for arthritis.

Although many studies have compared synovial fluid expression levels or protein to those of serum samples to look for corresponding targets to be exploited as biomarker profiles, it may be unlikely that only one or two proteins demonstrate reliable specificity with OA or RA. Using the methodology outlined in this study, the relationship between expression levels of MMPs can be merged into a single visual plot which allows for the reliable and reproducible identification of normal from diseased joints, however, this methodology cannot distinguish between normal and early OA synovial fluid, suggesting that the ‘degradative snap shot’ of the early OA joint may not be significantly different from that of a healthily joint at the level of the MMPs analyzed.