Institution: The University of Manchester
Investigators: Dr Sarah Heathfield, Professor Judith Hoyland
Stream: Research Fellowship
Topic: Lower Limbs (Hip)
Status: Completed
We have previously shown that in the degenerate intervertebral disc (IVD) there is tissue specific cellular senescence which adversely influences the ability of the IVD to repair itself. The purpose of this one year study, funded by the Furlong Foundation, was to examine the role of local environment factors (e.g. IL-1, mechanical load), which are altered in the degenerate IVD, in inducing the senescent cell phenotype that we have previously described. As such these could be key targets for future therapeutic interventions in the management of low back pain.
Aim 1: Examination of whether insults implicated in IVD degeneration (e.g. mechanical load and the cytokine IL-1) will induce cellular senescence.
We examined the effect of two local IVD environmental factors, namely mechanical load and the cytokine IL-1 on inducing/stimulating IVD cell senescence.
A) Does mechanical load induce senescence? The IVD is exposed to a variety of mechanical forces which may influence cell function, principally matrix homeostasis. Experiments were conducted to ascertain whether IVD cells exposed to cyclic stretch would undergo cell senescence. Cells were subjected to 10% strain at 0.3Hz or 1.0Hz for 20 minutes using a Flexercell strain device and gene expression analysed. Although mechanical load was seen to have an effect on matrix gene expression no changes in gene expression indicative of cell senescence were observed. Thus it appears that in the IVD exposure to abnormal mechanical load does not result in cell senescence.
B) Does IL-1 induce senescence? Much of the work undertaken throughout this year has focused on the possible role of the cytokine IL-1 in inducing senescence in human intervertebral disc cells. In vitro studies were conducted in which human IVD cells (from 5 different patient samples) were exposed to10ng/ml IL-1 for 24 hours and then assessed for markers of senescence. Treatment with IL-1 resulted in increased senescence associated B gal staining and cells showed an altered morphology. With regards to cell cycle progression a senescent cell population would be expected to accumulate in G1 or G2 phase of cell cycle and number of cells in S phase would be reduced. IL-1 treatment caused a 28% ± 5% reduction in number of cells in S phase, compared to untreated control, 24h after the start of treatment suggesting that IL-1beta causes cells to undergo cell cycle arrest and prevent cell proliferation (as evidenced by reduced Ki67[a marker of cell proliferation] staining). Associated with these changes was an increase in gene expression for caveolin 1 and the cell cycle inhibitor p21. The results suggest that IL-1 induces stress-induced premature senescence in human NP cells and that p21 and caveolin-1 are possible mediators of this change in cell behaviour. This data extends the importance of IL-1beta in IVD degeneration. The work is currently being written up for manuscript submission.
Aim 2: Reversing and Inducing senescence in IVD cells by adenoviral transfections.
This part of the study aimed to look at i) whether senescence can be reversed by genetically manipulating cells to over express hTERT, or down regulate molecules associated with senescence (e.g. caveolin 1, p16INK4a)., and ii) whether senescence could be induced through over-expression of caveolin -1 in order to study the effect of senescent cells on non-senescent cells. Standard operating procedures have been prepared and the necessary university documents completed to allow these experiments to be performed with in the laboratory.
Preliminary experiments involving optimisation of complex methodology have been undertaken to transfect cells with caveolin 1 in order to induce a senescent phenotype. Caveolin-1 protein overexpression was established in two degenerate NP cell samples but results to date has not shown conclusively that caveolin -1 overexpression leads to a senescent phenotype. Unfortunately time did not allow for further study and we are currently in the process of applying for further grant funding.
At the current time there is no definitive marker of a senescent cell and our studies (in contrast to other investigators who primarily utilise senescence associated beta galactosidase) utilise a number of potential markers to investigate the senescent cell phenotype. In order to aid identification of this cell state we have also began to investigate alternative potential markers. Work has been undertaken to investigate the expression of Cyclin D1 and DNA damage proteins in IVD as potential senescent markers. The work has demonstrated increased levels of Cyclin D1 protein with IVD degeneration and Cyclin D1 gene expression with increasing passage, suggesting a possible role for Cyclin D1 in replicative senescence in IVD cells . H2AXg, a component of DNA damage foci and downstream phosphorylation of kinase Chk2 were also found to be increased with disc degeneration and correlated with the expression of senescent markers.
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Mechanisms Underlying Cellular Sentence in Degeneration of the Intervertebral Disc: Implications for Novel Repair Strate
