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Title: The effects of mechanical stimulation and cytokine stimulation on proteoglycan levels in human chondrocytes
Author: Johnston, J. E.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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Transformed chondrocyte cell lines and primary OA human articular chondrocytes (HAC) were grown in monolayer culture. Cells were exposed to cyclical mechanical stimulation (MS) using an apparatus that produces strain on the base of the culture dish, causing the deformation of attached cells. Chondrocytes were also incubated with a cytokine cocktail containing IL-1β, TNFα, IL-6 and IFNγ (CYT). The transformed chondrocyte cell lines C20A4 and C2812 did not express detectable levels of NOS protein or nitrite activity. However increased inducible NOS (iNOS) mRNA following CYT stimulation suggested that the cells were able to sense the CYT stimulation. Primary OA HAC showed increased production of iNOS mRNA, protein and nitrite following CYT or IL-1β stimulation. The simultaneous application of CYT and MS also showed elevated iNOS mRNA, protein and nitrite levels, although these were significantly lower than following CYT alone. An IL-4 neutralising antibody and a β1 integrin function blocking antibody did not reverse the decreased CYT induced iNOS associated with MS. This suggests that MS decreases CYT induced iNOS through an IL-4 and a β1 integrin independent mechanism. A novel iNOS inhibitor (AR-C102222) was shown to have little effect on iNOS mRNA and protein levels following CYT stimulation of primary OA HAC. However, a dose dependent decrease in nitrite production was seen. Aggrecan mRNA levels in primary OA HAC were decreased following stimulation with CYT. The possible role of NO in this decreased aggrecan mRNA level was investigated. MS and a novel iNOS inhibitor both decreased the production of NO, but did not alter the CYT mediated decreased aggrecan mRNA. The NO donor SNAP did not alter aggecan mRNA levels. These results suggest that NO is not involved in regulating aggrecan mRNA levels. The final study using cells from an iNOS knockout mouse suggested that both NO dependent and independent mechanisms exist by which CYT can decrease aggrecan mRNA levels.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available