C.R. Flannery

Changes in serum and synovial fluid biomarkers after acute injury (NCT00332254)

IntroductionAcute trauma involving the anterior cruciate ligament is believed to be a major risk factor for the development of post-traumatic osteoarthritis 10 to 20 years post-injury. In this study, to better understand the early biological changes which occur after acute injury, we investigated synovial fluid and serum biomarkers.MethodsWe collected serum from 11 patients without pre-existing osteoarthritis from a pilot intervention trial (5 placebo and 6 drug treated) using an intra-articular interleukin-1 receptor antagonist (IL-1Ra) therapy, 9 of which also supplied matched synovial fluid samples at presentation to the clinic after acute knee injury (mean 15.2 ± 7.2 days) and at the follow-up visit for reconstructive surgery (mean 47.6 ± 12.4 days). To exclude patients with pre-existing osteoarthritis (OA), the study was limited to individuals younger than 40 years of age (mean 23 ± 3.5) with no prior history of joint symptoms or trauma. We profiled a total of 21 biomarkers; 20 biomarkers in synovial fluid and 13 in serum with 12 biomarkers measured in both fluids. Biomarkers analyzed in this study were found to be independent of treatment (P > 0.05) as measured by Mann-Whitney and two-way ANOVA.ResultsWe observed significant decreases in synovial fluid (sf) biomarker concentrations from baseline to follow-up for sfC-Reactive protein (CRP) (P = 0.039), sflubricin (P = 0.008) and the proteoglycan biomarkers: sfGlycosaminoglycan (GAG) (P = 0.019), and sfAlanine-Arginine-Glycine-Serine (ARGS) aggrecan (P = 0.004). In contrast, we observed significant increases in the collagen biomarkers: sfC-terminal crosslinked telopeptide type II collagen (CTxII) (P = 0.012), sfC1,2C (P = 0.039), sfC-terminal crosslinked telopeptide type I collagen (CTxI) (P = 0.004), and sfN-terminal telopeptides of type I collagen (NTx) (P = 0.008). The concentrations of seven biomarkers were significantly higher in synovial fluid than serum suggesting release from the signal knee: IL-1β (P < 0.0001), fetal aggrecan FA846 (P = 0.0001), CTxI (P = 0.0002), NTx (P = 0.012), osteocalcin (P = 0.012), Cartilage oligomeric matrix protein (COMP) (P = 0.0001) and matrix metalloproteinase (MMP)-3 (P = 0.0001). For these seven biomarkers we found significant correlations between the serum and synovial fluid concentrations for only CTxI (P = 0.0002), NTx (P < 0.0001), osteocalcin (P = 0.0002) and MMP-3 (P = 0.038).ConclusionsThese data strongly suggest that the biology after acute injury reflects that seen in cartilage explant models stimulated with pro-inflammatory cytokines, which are characterized by an initial wave of proteoglycan loss followed by subsequent collagen loss. As the rise of collagen biomarkers in synovial fluid occurs within the first month after injury, and as collagen loss is thought to be irreversible, very early treatment with agents to either reduce inflammation and/or reduce collagen loss may have the potential to reduce the onset of future post-traumatic osteoarthritis.Trial registrationThe samples used in this study were derived from a clinical trial NCT00332254 registered with ClinicalTrial.gov.

Elevated aggrecanase activity in a rat model of joint injury is attenuated by an aggrecanase specific inhibitor

OBJECTIVE To evaluate aggrecanase activity after traumatic knee injury in a rat model by measuring the level of aggrecanase-generated Ala-Arg-Gly-aggrecan (ARG-aggrecan) fragments in synovial fluid, and compare with ARG-aggrecan release into joint fluid following human knee injury. To evaluate the effect of small molecule inhibitors on induced aggrecanase activity in the rat model. METHOD An enzyme-linked immunosorbent assay (ELISA) was developed to measure ARG-aggrecan levels in animal and human joint fluids. A rat model of meniscal tear (MT)-induced joint instability was used to assess ARG-aggrecan release into joint fluid and the effects of aggrecanase inhibition. Synovial fluids were also obtained from patients with acute joint injury or osteoarthritis and assayed for ARG-aggrecan. RESULTS Joint fluids from human patients after knee injury showed significantly enhanced levels of ARG-aggrecan compared to uninjured reference subjects. Similarly, synovial fluid ARG-aggrecan levels increased following surgically-induced joint instability in the rat MT model, which was significantly attenuated by orally dosing the animals with AGG-523, an aggrecanase specific inhibitor. CONCLUSIONS Aggrecanase-generated aggrecan fragments were rapidly released into human and rat joint fluids after injury to the knee and remained elevated over a prolonged period. Our findings in human and preclinical models strengthen the connection between aggrecanase activity in joints and knee injury and disease. The ability of a small molecule aggrecanase inhibitor to reduce the release of aggrecanase-generated aggrecan fragments into rat joints suggests that pharmacologic inhibition of aggrecanase activity in humans may be an effective treatment for slowing cartilage degradation following joint injury.

Tenascin-C induces inflammatory mediators and matrix degradation in osteoarthritic cartilage

BackgroundTenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed.MethodsTN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA.ResultsTN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE2, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA.ConclusionsTN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.

Serine Protease HTRA1 Antagonizes Transforming Growth Factor-β Signaling by Cleaving Its Receptors and Loss of HTRA1 In Vivo Enhances Bone Formation

HTRA1 is a member of the High Temperature Requirement (HTRA1) family of serine proteases, which play a role in several biological and pathological processes. In part, HTRA1 regulation occurs by inhibiting the TGF-β signaling pathway, however the mechanism of inhibition has not been fully defined. Previous studies have shown that HTRA1 is expressed in a variety of tissues, including sites of skeletal development. HTRA1 has also been implicated in the process of bone formation, although the precise manner of regulation is still unknown. This study investigated how HTRA1 regulates TGF-β signaling and examined the in vivo effects of the loss of HTRA1. We demonstrated that recombinant HTRA1 was capable of cleaving both type II and type III TGF-β receptors (TβRII and TβRIII) in vitro in a dose-dependent manner, but it did not affect the integrity of TβRI or TGF-β. Overexpression of HTRA1 led to decreased levels of both TβRII and III on the cell surface but had no effect on TβRI. Silencing HTRA1 expression significantly increased TGF-β binding to the cell surface and TGF-β responsiveness within the cell. To examine the role of HTRA1 in vivo, we generated mice with a targeted gene deletion of HTRA1. Embryonic fibroblasts isolated from these mice displayed an increase in TGF-β-induced expression of several genes known to promote bone formation. Importantly, the loss of HTRA1 in the knockout mice resulted in a marked increase in trabecular bone mass. This study has identified a novel regulatory mechanism by which HTRA1 antagonizes TGF-β signaling, and has shown that HTRA1 plays a key role in the regulation of bone formation.

Not All Lubricin Isoforms Are Substituted with a Glycosaminoglycan Chain

Lubricin, also referred to as superficial zone protein, has been reported to be a proteoglycan. However, the structure of its glycosaminoglycan chain has not been well characterized, and this study was undertaken to investigate the structure of the glycosaminoglycan chain that decorated lubricin in human synovial fluid to provide insight into its biological role. Lubricin was detected as a major band at approximately 360 kDa which co-migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a chondroitin sulfate (CS)-containing proteoglycan that was detected by both monoclonal antibodies (MAb) 2-B-6 and MAb 3-B-3 after chondroitinase ABC treatment and keratan sulfate (KS) that was detected by MAb 5-D-4. Further analysis of lubricin-containing fractions that eluted from an anion exchange column indicated that the major population of lubricin could be separated from the CS and KS stubs which indicated that this fraction of lubricin was not decorated with glycosaminoglycan chain and was the glycoprotein form of lubricin. Lubricin present in fractions that also contained CS was found to be decorated with CS structures which were reactive with MAb 3-B-3 after chondroitinase ABC digestion using a sandwich enzyme-linked immunosorbent assay approach. Aggrecan was not found to form complexes with lubricin in synovial fluid which confirmed that the MAb 3-B-3 CS and MAb 5-D-4 KS structures decorated lubricin. These data demonstrate that lubricin present in human synovial fluid was a heterogeneous population with both glycoprotein and proteoglycan forms.

Lubricin Its Presence in Repair Cartilage following Treatment with Autologous Chondrocyte Implantation

Objective: To determine if lubricin was present in the surface layer of repair cartilage formed after autologous chondrocyte implantation (ACI). Design: Forty-three biopsies of repair tissue were taken from patients who had been treated with ACI 8 to 68 months previously (mean of 18.0 ± 14.4 months); 30 had flaps of periosteum and 13 of Chondro-Gide®. Cryopreserved sections were stained with hematoxylin and eosin, toluidine blue, and immunostained for lubricin and type II collagen. The quality of repair tissue was scored via OsScore, and clinical improvement in patients was assessed via change in Lysholm score. Normal/control cartilage was studied for comparison (n = 5). Results: Patients’ Lysholm scores improved from 48.1 ± 17 preoperatively to 69.5 ± 21.5 posttreatment. The thickness of repair tissue was 2.9 ± 1.7 mm compared with 2.3 ± 0.6 mm for control cartilage, with an OsScore of 6.7 ± 1.6 (8.9 ± 1.2 for controls). Ninety-eight percent of biopsies had staining for lubricin, with 84% having some in the surface layer (60% of periosteal treated and 100% of Chondro-Gide treated). The improvement in Lysholm score was not significantly different in patients with lubricin present at the surface compared with those without. Conclusion: Lubricin was present in almost all samples of repair tissue formed post ACI, often in the surface layer, resembling the distribution that is seen in normal cartilage. The presence of lubricin in the upper layer is likely to have implications for the functioning of the tissue because, via its mucin-like repeats, it appears capable of reducing the friction that could arise in articulating joints.

143 BENEFICIAL EFFECTS OF LIVER X RECEPTOR (LXR) MODULATION ON MATRIX METABOLISM AND PROSTAGLANDIN E2 (PGE2) PRODUCTION IN CARTILAGE

were treated with H-89, a PKA inhibitor, W-7, a calmodulin inhibitor or LY294002, a PI3K inhibitor prior to 4a-PDD stimulation in SOX9dependent reporter assay. To examine the chondrogenic differentiation, ATDC5 cells were co-stimulated with 0 to 120 nM 4a-PDD and 10mg/ml insulin for 10 day, then cells were stained with alcian blue. The amount of SOX9 protein was estimated by Western Blot analysis using anti-SOX9 antibody. Results: We have screened 120,000 cDNA clones and identified 46 genes that activated SOX9-dependent reporter activity. In cDNA microarray analysis, the mRNA levels of Sox5, Sox6, Ifitm5, Myd116, Mef2c and TRPV4 genes were elevated during chondrogenic differentiation of ATDC5 cells treated with insulin. TRPV4, a cation channel molecule was further investigated in this study since it had a strong effect on SOX9dependent transcription. mRNA expression of TRPV4 gene was observed in ATDC5, C3H10T1/2, murine primary chondrocytes prepared from the rib cages and hind limb buds in embryonic day12 embryos, but not in NIH3T3. When ATDC5 cells or C3H10T1/2 cells were treated with various concentration of 4a-PDD, SOX9-dependent transcription was elevated in dose dependent manner and this effect was abolished by the addition of ruthenium red, a TRPV antagonist. In ATDC5 cells, H-89 and W-7 inhibited SOX9-dependent reporter activity caused by the stimulation with 4a-PDD while LY294002 did not. When ATDC5 cells were co-stimulated with 4a-PDD and insulin, GAG accumulation was significantly increased as compared with insulin alone whereas 4a-PDD alone showed no effect. Similar result was obtained in C3H10T1/2 cells co-stimulated with 4aPDD and BMP-2. Co-stimulation with 4a-PDD demonstrated further elevations of mRNAs for type II collagen and aggrecan in ATDC5 cells when compared to insulin alone. 4a-PDD stimulation increased the amount of SOX9 protein in both ATDC5 and C3H10T1/2 cells. Conclusions: We have identified TRPV4 by its ability to activate SOX9dependent transcription. Activation of TRPV4 promoted chondrogenic differentiation of ATDC5 cells in cooperation with insulin and C3H10T1/2 cells in corporation with BMP-2 in vitro. The protein level of SOX9 was increased by the stimulation with 4a-PDD. These observations suggest that TRPV4 may correlate the process of chondrogenesis.

A13 INTRAARTICULAR LUBRICIN SUPPLEMENTATION IS CHONDROPROTECTIVE AND PREVENTS CARTILAGE DEGENERATION IN EXPERIMENTAL OSTEOARTHRITIS

12 weeks post-BTM while Group C were sacrificed 12 [n = 6], 24 [n = 6] and 52 [n = 6] weeks post-BTM. At necropsy, both medial compartments of BTM joints were scored by 2 blinded observers for AC lesions and osteophytes (OP) using a 0−4 scale. Synovial tissue and a 5mm wide coronal osteochondral slice were removed from the mid-line of the femur and tibia and processed for histochemical and histomorphometric analyses using published methods. Intact patellae were used for topographical biomechanical AC indentation studies. Results: Gross morphological scores 12 wks post BTM showed a dosedependent effect of MPC on AC integrity and OP formation; 100 mil MPC emerging as the most effective chondroprotective dose relative to HA alone. Total AC score ratios (HA+MPC)/(HA) showed 100>150>25=10 while OP ratios were 100>25>10>150 mil MPC. Statistically significant (SS) lower scores were observed for total femoral & tibial AC (p = 0.019) and total AC and OP (p = 0.009) for Group C MPC joints compared HA alone. Histomorphometric analysis of Group C MPC+HA tibial plateaus revealed that AC were thicker than the corresponding HA-AC in the middle (p = 0.057) and outer regions (p = 0.028); for all regions (p = 0.01). The mean phase lag for the patellae AC of Group C MPC injected joints was significantly lower than the contralateral patella AC (p = 0.002). Mean modified Mankin scores for AC sections from Group C MPC+HA joints were less than corresponding HA sections but were not SS. There was no evidence of synovial histopathology modulation. The chondroprotective effects observed for the 100 mil MPC injected joints diminished with time; the positive effects noted at 12 and 24 weeks BTM being lost by 52 weeks. Conclusions: This is the first report, as far as we are aware, of a beneficial therapeutic effect of allogenic Stro-3+ MPC on cartilage integrity in a model of early OA. MPC/MSC are known to release growth factors and cytokines and also suppress the production of TNF-alpha by other cells, while up-regulating anti-inflammatory cytokines (eg. Il-4, Il-10). These paracrine activities of MPC could stimulate chondrocyte biosynthesis of new matrix but also attenuate local production and activity of catabolic mediators. The finding in this study that 100 million MPC were chondroprotective was consistent with such a mechanism of action.

15.3 Immunolocalisation of Lubricin (SZP) in biopsies of ACI-treated patients

Immunolocalisation of Lubricin (SZP) in biopsies of ACI-treated patients S. Roberts, J.B. Richardson, J. Menage, E.H. Evans, A.R. Jones, C.R. Flannery; Centre For Spinal Studies, Robert Jones & Agnes Hunt Orthopaedic Hospital, Oswestry, United Kingdom, Institute Of Orthopaedics, RJAH Orthopaedic Hospital, Shropshire, United Kingdom, Centrre For Spinal Studies, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire, United Kingdom, Centre For Spinal Studies, RJAH Orthopaedic Hospital, Shropshire, United Kingdom, Wyeth Research, Wyeth Research, Cambridge, MA, United States of America