Urocortin protects chondrocytes from acute trauma in articular cartilage : WestminsterResearch

Title	Urocortin protects chondrocytes from acute trauma in articular cartilage
Authors	Jones, R.C., Jackson, TR, Locke, I.C., Board, T.N., Richardson, S.M., Townsend, P.A. and Lawrence, K.M.
Type	Conference poster
Abstract	Purpose: Single injurious mechanical impact to joints, as seen in sports injury, is known to be associated with a high risk of later development of osteoarthritis (OA). Irreversible chondrocyte death is initiated following impact and results in progressive breakdown of articular cartilage. Promotion of chondrocyte survival is therefore a necessary target in the prevention of development of further OA. Urocortin1 (Ucn) has recently been shown to have a pro-survival effect on chondrocytes in both the presence and absence of pro-apoptotic stimuli. The aim of this study was to investigate the role of Ucn in response to mechanical impact. Methods: Full thickness porcine cartilage explants from lateral and medial femoral condyles were impacted with a 500g weight using a custom-made drop tower device. Impacted explants were treated with human Ucn (10-7M, 30 minutes prior to or following impact), non-selective cation channel blocker Gadolinium (500μM, 30 minutes following impact), or mechanosensitive ion channel blocker GsMTx4 (40μM, 2 hours prior to impact) and cultured for 72hrs. Cell viability was assessed with a CMFDA:PI live dead assay, using both macro confocal microscopy, as a means of avoiding bias, and high magnification confocal microscopy techniques. Lactate dehydrogenase (LDH) assays were conducted at 72hrs to provide a quantitative read out of cell death for each explant. Calcium influx was assessed 1hr post impact by Fluo-4 AM staining and visualised by multiphoton microscopy. Results: Porcine explants were impacted from a range of heights (10-70mm) in order to establish the correct energy of impact for inducing cell death without excess damage and fracture of the cartilage explant. Cell death increased in a dose dependent manner with height. 50mm was chosen as optimal height for all further experiments. Pre-treatment of explants with Ucn had a significant increase on cell viability when compared to impacted alone, as seen by quantification of the CMFDA:PI ratio on macro confocal (p=0.0042) and by high magnification confocal microscopy (p=0.0015). This increase in cell viability was confirmed by LDH assay (p=0.0063). Post-impact treatment of the explants with Ucn also resulted in a significant increase in cell survival, seen in both CMFDA:PI ratio (p=0.0009) and LDH assay (p=0.0053). We have previously found in primary human chondrocytes that Ucn exerts its protective effect on chondrocytes by the inhibition of excessive calcium influx. Calcium influx was therefore visualised in impacted explants treated pre- and post-impact with Ucn. A significant decrease in the levels of calcium staining was seen in both of these conditions when compared to explants impacted in the absence of Ucn (p=0.0073, p=0.05). In vitro work also highlighted a role for the mechanosensitive ion channel Piezo1 in cell death associated with the absence of Ucn. Explants were therefore treated with both Gadolinium and GsMTx4 post-impact and cell viability assessed by multiphoton microscopy. An increase in cell viability was observed in both conditions, comparable to those seen with addition of Ucn. Conclusions: A novel role for Ucn in the protection of articular chondrocytes maintained in cartilage explants has been shown and validated by two microscopy techniques and LDH assay. Ucn has also been shown to reduce calcium influx in impacted explants at 60 minutes post impact, and increased cell viability was observed by blocking mechanosensitive ion channels. These data suggest the importance of regulation of calcium influx in Ucn-mediated cell protection. The pro-survival effect of Ucn was seen both pre- and post-impact, highlighting the potential prophylactic and therapeutic use of Ucn for treating sudden injury to cartilage, and subsequent progression to post-traumatic OA.
Year	2017
Conference	OARSI 2017 World Congress on Osteoarthritis
Publisher	Elsevier
Journal	Osteoarthritis and Cartilage
Journal citation	25, pp. 169-170
ISSN	1063-4584
Digital Object Identifier (DOI)	https://doi.org/10.1016/j.joca.2017.02.291

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Prashar, A., Veness, R., Locke, I.C. and Evans, C.S. 2002. Effect of essential oils and their components on cell. Future Trends in Phytochemistry. Gargnano, Italy

Varidase: the science behind the medicament
Rutter, P.M., Carpenter, B.G., Hill, S.S. and Locke, I.C. 2000. Varidase: the science behind the medicament. Journal of Wound Care. 9 (5), pp. 223-226.

A comparison of the characteristics of circulating anti-myeloperoxidase antibodies in vasculitis with those in non-vasculitic conditions
Locke, I.C., Leaker, B. and Cambridge, G. 1999. A comparison of the characteristics of circulating anti-myeloperoxidase antibodies in vasculitis with those in non-vasculitic conditions. Clinical and Experimental Immunology. 115 (2), pp. 369-376. https://doi.org/10.1046/j.1365-2249.1999.00809.x

A comparison of the variable region genes of two human monoclonal antibodies to myeloperoxidase
Locke, I.C., Rahman, M.A.A. and Cambridge, G. 1998. A comparison of the variable region genes of two human monoclonal antibodies to myeloperoxidase. British Society for Immunology 6th Annual Meeting. Harrogate, UK 12/1998

Adjacent proline residues in the inhibitory domain of the Oct-2 transcription factor play distinct functional roles
Liu, Y.Z., Lee, I.K., Locke, I.C., Dawson, S. and Latchman, D. 1998. Adjacent proline residues in the inhibitory domain of the Oct-2 transcription factor play distinct functional roles. Nucleic Acids Research. 26 (10), pp. 2464-2472. https://doi.org/10.1093/nar/26.10.2464

Purification of a streptococcal deoxyribonuclease by affinity chromatography based on a DNA-cellulose matrix
Locke, I.C., Cox, S.F. and Carpenter, B.G. 1997. Purification of a streptococcal deoxyribonuclease by affinity chromatography based on a DNA-cellulose matrix. Journal of Chromatography A. 788 (1-2), pp. 75-80. https://doi.org/10.1016/S0021-9673(97)00695-X

Autoantibodies to myeloperoxidase in systemic sclerosis
Locke, I.C., Worral, J.G., Leaker, B., Black, C.M. and Cambridge, G. 1997. Autoantibodies to myeloperoxidase in systemic sclerosis. Journal of Rheumatology. 24 (1), pp. 86-89.

Characteristics of antibodies to myeloperoxidase in vasculitic and non-vasculitic conditions
Locke, I.C., Leaker, B. and Cambridge, G. 1996. Characteristics of antibodies to myeloperoxidase in vasculitic and non-vasculitic conditions. American College of Rheumatology 60th National Scientific Meeting. Orlando, USA 10/1996

Characteristics of anti-myeloperoxidase autoantibodies in vasculitis and other conditions
Locke, I.C., Leaker, B. and Cambridge, G. 1996. Characteristics of anti-myeloperoxidase autoantibodies in vasculitis and other conditions. American Society of Nephrology 29th Annual Meeting. New Orleans, USA 11/1996

Autoantibodies to neutrophil granule proteins: pathogenic potential in vasculitis?
Locke, I.C. and Cambridge, G. 1996. Autoantibodies to neutrophil granule proteins: pathogenic potential in vasculitis? British Journal of Biomedical Science. 53 (4), pp. 302-316.

A human monoclonal anti-myeloperoxidase antibody can prime human neutrophils in vitro
Cambridge, G., Leaker, B., Locke, I.C., Noronha-Dutra, A. and Epperlein, M. 1996. A human monoclonal anti-myeloperoxidase antibody can prime human neutrophils in vitro. American Society of Nephrology 29th Annual Meeting. New Orleans, USA 11/1996

Autoantibodies to myeloperoxidase in systemic sclerosis
Worral, J.G., Locke, I.C., Leaker, B., Black, C.M. and Cambridge, G. 1995. Autoantibodies to myeloperoxidase in systemic sclerosis. British Society for Rheumatology Heberden Round 1995. Keele University, UK 9/1995

An automated method for the determination of deoxyribonuclease activity as exemplified by fractionation of the components of the medicament Varidase®
Locke, I.C., Ramsay, M.P., Hill, S.S. and Carpenter, B.G. 1993. An automated method for the determination of deoxyribonuclease activity as exemplified by fractionation of the components of the medicament Varidase®. The Journal of Automatic Chemistry. 15 242749.

Automated analysis of the deoxyribonuclease active constituents of the medicament Varidase ®
Locke, I.C., Ramsey, M.P., Hill, S.S. and Carpenter, B.G. 1992. Automated analysis of the deoxyribonuclease active constituents of the medicament Varidase ®. IMLS XX Triennial Conference. Liverpool, UK 9/1992

Purification of the seed lectin from Dolichos biflorus by chromatofocusing
Rogers, D.J., Locke, I.C., Gibbs, R. and Carpenter, B.G. 1990. Purification of the seed lectin from Dolichos biflorus by chromatofocusing. 12th International Lectin Conference. University of Davis, USA 9/1990

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Urocortin protects chondrocytes from acute trauma in articular cartilage

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