Periodontal ligament inflammation or periodontitis is a common disease characterised by gradual destruction of connective tissue fibres that attach a tooth to the alveolar bone within which it sits. Diabetes and inflammation enhances periodontal bone loss through enhanced resorption and diminished bone formation. Periodontal ligament fibroblast attachment to collagen-I and function was impaired by methylglyoxal (MG) modification in vitro. The glyoxalase system is an anti-glycation defence in all cells that metabolises MG and thereby suppresses MG-mediated protein damage. Overexpression of Glo1 decreased the intracellular levels of MG The aim of this investigation was to improve the understanding of protein damage in PDL in diabetes, focusing on protein damage by MG in human periodontal ligament fibroblasts (hPDLFs) in hyperglycaemia and to evaluate the effects of high and low glucose concentrations on MG metabolism in hPDLFs with or without Glo1 inducers. The effect of high glucose concentration on the formation and metabolism of MG was studied in hPDLFs in vitro. The ability of two small molecule Glo1 inducers, individually and in synergistic combination, to counter dicarbonyl stress in hPDLFs in vitro was studied. Interactions between hPDLFs to the extracellular matrix protein, collagen-I, were investigated and impairments in hPDLFs adhesion to MG-modified collagen-I coated plates were assessed. Protein susceptible to MG modification and inactivation in the cytosol of hPDLFs were identified by high resolution mass spectrometry proteomics. The effect of clinical periodontitis on plasma protein glycation, oxidation and nitration was also investigated in a pilot clinical investigation. When hPDLFs were incubated with high glucose concentration in vitro there was a 45% decrease in Glo1 activity and 42% increase in D-lactate flux – surrogate indication of MG flux of formation, which contributed to increased cellular concentration of MG and increase in MG-H1 residue content of cell protein, compared to low glucose control. This indicated dicarbonyl stress was induced in hPDLFs by high glucose concentration in vitro, a model for hyperglycaemia in vivo. Decrease of Glo1 activity and increase in cellular MG concentration and MG-H1 residue content of cell protein was corrected with the addition of Glo1 inducers. The binding of hPDLFs to collagen-I was decreased by 30% in high glucose concentration and was corrected by addition of Glo1 inducers. Proteomics analysis of cytosolic extracts of hPDLFs indicated that high glucose incubations produced changes in MG-modified proteins and also up-regulated and down-regulated unmodified proteins in hPDLFs. The pilot investigation of clinical periodontitis suggested a systemic effect of this local inflammation which was associated with changes in plasma protein glycation, oxidation and nitration. This study reveals that dicarbonyl stress is a potential contributory pathogenic mechanism in hPDLFs in periodontitis and countering it may provide new treatment options to prevent and treat decline in periodontal health, particularly in diabetes. Small molecule inducers of Glo1 expression may in future contribute to improving periodontal health, particularly in diabetes.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:690496 |
| Date | January 2016 |
| Creators | Ashour, Amal |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/80026/ |
Page generated in 0.0024 seconds