THU0435 CALCIUM PYROPHOSPHATE DIHYDRATE (CPPD) CRYSTALS BUT NOT BASIC CALCIUM PHOSPHATE (BCP) CRYSTALS INDUCE SYNDECAN-4 EXPRESSION IN CARTILAGE

Background:Chondrocalcinosis is a painful rheumatic condition caused by the deposition of calcium pyrophosphate dihydrate crystals (CPPD) in joint tissues, and especially in cartilage. It is known that CPPD crystals cause inflammation and degenerative changes in joint, but the underlying mechanisms remain poorly understood. In particular, nothing is known about how these crystals regulates transmembrane heparan sulphate proteoglycans (HSPGs). Our attention focused on one family of HSPGs called syndecans as they have important roles both as adhesion molecules, by mediating chondrocyte-extracellular matrix interactions, and as modulators of intracellular signaling triggered by cytokines and growth factors.Objectives:The aim of this study was to evaluate how CPPD crystals modulates syndecan expression in chondrocytes and in cartilage, and how this modulation can be ultimately linked to cartilage damage during chondrocylcinosis.Methods:Murine chondrocitic ATDC5 cells were stimulated with 0,1 ng/ml CPPD crystals or with 0,1 ng/ml basic-calcium phosphate crystals (BCP), a family of calcium-containing crystals found in other rheumatic conditions such as osteoarthritis (OA). Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release in the supernatant at 30 minutes, and 3, 6, 24 hours after stimulation. At the same time-points, mRNA expression levels of syndecans (Synd-1, -2, -3, -4) and of matrix-degrading enzymes (Mmp-3, -9, -13; Adamts-4, -5) was analysed by qRT-PCR. Finally, Syndecan-4 protein expression was studied by immunohistochemistry (IHC) in cartilage samples of patients with chondrocalcinosis and in samples of patients with severe OA without chondrocalcinosis as control.Results:LDH assay revealed no increased cytotoxicity by CPPD or BCP at any time-point. qRT-PCR indicated that CPPD crystals but not BCP crystals induced Synd-2 and -3 upregulation at 30 minutes after stimulation and Synd-4 upregulation at 3 hours, while no modulation of syndecans was seen at later time-points. CPPD also induced Adamts-4 expression at 3 hours after stimulation, and Mmp-9 expression at 3 and 6 hours. The expression of the other matrix-degrading enzymes was not affected. Human chondrocalcinosis cartilage exhibited enhanced Synd-4 expression compared to severe OA cartilage containing BCP calcification. Interestingly, Synd-4 expression was observed in the extracellular matrix but not on cell membrane, suggesting that maybe Synd-4 undergoes shedding (Figure 1).Figure 1.Representative Synd-4 IHC in control patients (Ctrl, severe OA) and chondrocalcinosis patients (CPPD). Note increased Synd-4 expression in extracellular matrix of CPPD patients compared to Ctrl ones.Conclusion:BCP and CPPD crystals seem to trigger differential effects in terms of modulation of syndecans in chondrocitic cells. CPPD crystals induce Synd-4 and Adamts-4 and Mmp-9 which are not induced by BCP crystals. It remains to be clarified whether the two events are interlinked. In particular, further studies are required to investigate if Adamts-4 and Mmp-9 are involved in Synd-4 shedding or if vice versa Synd-4 regulates Adamts-4 and Mmp-9 activation and downstream cartilage breakdown in chondrocalcinosis.Disclosure of Interests: :Sonia Nasi: None declared, Jessica Bertrand Grant/research support from: Pfizer, Speakers bureau: Pfizer, Miriam Bollmann: None declared, Richard Stange: None declared, Thomas Pap: None declared

Dual-energy computed-tomography-based discrimination between basic calcium phosphate and calcium pyrophosphate crystal deposition in vivo

Background: Dual-energy computed tomography (DECT) is being considered as a non-invasive diagnostic and characterization tool in calcium crystal-associated arthropathies. Our objective was to assess the potential of DECT in distinguishing between basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) crystal deposition in and around joints in vivo. Methods: A total of 13 patients with calcific periarthritis and 11 patients with crystal-proven CPPD were recruited prospectively to undergo DECT scans. Samples harvested from BCP and CPP calcification types were analyzed using Raman spectroscopy and validated against synthetic crystals. Regions of interest were placed in BCP and CPP calcifications, and the following DECT attenuation parameters were obtained: CT numbers (HU) at 80 and 140 kV, dual-energy index (DEI), electron density (Rho), and effective atomic number ( Zeff). These DECT attenuation parameters were compared and validated against crystal calibration phantoms at two known equal concentrations. Receiver operating characteristic (ROC) curves were plotted to determine the highest accuracy thresholds for DEI and Zeff. Results: Raman spectroscopy enabled chemical fingerprinting of BCP and CPP crystals both in vitro and in vivo. DECT was able to distinguish between HA and CPP in crystal calibration phantoms at two known equal concentrations, most notably by DEI (200 mg/cm3: 0.037 ± 0 versus 0.034 ± 0, p = 0.008) and Zeff (200 mg /cm3: 9.4 ± 0 versus 9.3 ± 0, p = 0.01) analysis. Likewise, BCP calcifications had significantly higher DEI (0.041 ± 0.005 versus 0.034 ± 0.005, p = 0.008) and Zeff (9.5 ± 0.2 versus 9.3 ± 0.2, p = 0.03) than CPP crystal deposits with comparable CT numbers in patients. With an area under the ROC curve of 0.83 [best threshold value = 0.0 39, sensitivity = 90. 9% (81.8, 97. 7%), specificity = 64.6% (50.0, 64. 6%)], DEI was the best parameter in distinguishing between BCP and CPP crystal depositions. Conclusion: DECT can help distinguish between crystal-proven BCP and CPP calcification types in vivo and, thus, aid in the diagnosis of challenging clinical cases, and in the characterization of CPP and BCP crystal deposition occurring in osteoarthritis.

Crystal-induced musculoskeletal disease

This chapter describes crystal-induced musculoskeletal disease, which encompasses gout, hyperuricaemia, calcium pyrophosphate dihydrate disease, basic calcium phosphate-associated disease, and calcium oxalate arthritis. Their epidemiology, aetiopathogenesis, classification criteria, presenting features, clinical manifestations, and management are discussed. Diet, lifestyle, non-pharmacological and pharmacological approaches to their management are considered. User-friendly algorithms for the management of gout and calcium pyrophosphate dihydrate disease are provided, allowing both rheumatologists and non-rheumatologists to confidently manage these diseases in clinic, ward, or community settings.