Abstract
Intervertebral disc (IVD) cells derived from degenerate tissue respond aberrantly to mechanical stimuli, potentially due to altered mechanotransduction pathways. Elucidation of the altered, or alternative, mechanotransduction pathways operating with degeneration could yield novel targets for the treatment of IVD disease. Our aim here was to investigate the involvement of RGD-recognising integrins and associated signalling molecules in the response to cyclic tensile strain (CTS) of human annulus fibrosus (AF) cells derived from non-degenerate and degenerate IVDs. AF cells from non-degenerate and degenerate human IVDs were cyclically strained with and without function blocking RGD – peptides with 10% strain, 1.0 Hz for 20 minutes using a Flexercell® strain device. QRT-PCR and Western blotting were performed to analyse gene expression of type I collagen and ADAMTS -4, and phosphorylation of focal adhesion kinase (FAK), respectively. The response to 1.0 Hz CTS differed between the two groups of AF cells, with decreased ADAMTS -4 gene expression and decreased type I collagen gene expression post load in AF cells derived from non-degenerate and degenerate IVDs, respectively. Pre-treatment of non-degenerate AF cells with RGD peptides prevented the CTS-induced decrease in ADAMTS -4 gene expression, but caused an increase in expression at 24 hours, a response not observed in degenerate AF cells where RGD pre-treatment failed to inhibit the mechano-response. In addition, FAK phosphorylation increased in CTS stimulated AF cells derived from non-degenerate, but not degenerate IVDs, with RGD pre-treatment inhibiting the CTS – dependent increase in phosphorylated FAK. Our findings suggest that RGD -integrins are involved in the 1.0 Hz CTS – induced mechano-response observed in AF cells derived from non-degenerate, but not degenerate IVDs. This data supports our previous work, suggesting an alternative mechanotransduction pathway may be operating in degenerate AF cells.
Highlights
The intervertebral disc (IVD) is subject to a variety of mechanical stimuli, including compressive and tensile strains, hydrostatic pressures (HP) and fluid flow [1,2]
Gene Expression Analysis The genes chosen for analysis were based on previously published observations, whereby from a panel of extracellular matrix and matrix degrading enzyme genes investigated, only MMP -3 and ADAMTS -4, and aggrecan and type I collagen gene expression were altered in 1.0 Hz cyclic tensile strain (CTS) treated non-degenerate and degenerate annulus fibrosus (AF) cells, respectively [40]
ADAMTS -4 and type I collagen were chosen as the genes to be analysed, as these genes had shown the greatest fold changes in response to 1.0 Hz CTS of non-degenerate and degenerate AF cells, respectively [22]
Summary
The intervertebral disc (IVD) is subject to a variety of mechanical stimuli, including compressive and tensile strains, hydrostatic pressures (HP) and fluid flow [1,2]. We have recently shown that the reduction in catabolic gene expression observed in human AF cells derived from non-degenerate IVDs subjected to a physiologically relevant stimulus of 1.0 Hz cyclic tensile strain (CTS), is replaced by a reduction in anabolic gene expression in cells derived from a degenerate disc [12]. Such findings suggest that physiological mechanical stimuli important for matrix homeostasis could, become detrimental with IVD degeneration resulting in aberrant cell responses and potentially leading to progression of DDD. If the mechanotransduction pathways are found to be altered in disc cells derived from degenerate tissue, elucidation of the pathways could lead to an improved understanding of the aetiology of DDD and potentially to the discovery of novel therapeutic targets for the prevention, and/or treatment, of IVD degeneration
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