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BS59 Loss of SIRT1 in diabetes enhances dna damage and reduces MRN complex activation resulting in increased vascular calcification
  1. Francesca Bartoli-Leonard,
  2. Yvonne Alexander,
  3. Ria Weston
  1. Manchester Metropolitan University

Abstract

Introduction Patients with type II diabetes have considerably higher risk of developing cardiovascular disease and often have an elevated prevalence of vascular calcification combined with a loss of SIRT1, which induces osteogenic differentiation within the smooth muscle. This study aims to determine whether the loss of SIRT1 contributes to the enhanced DNA damage associated with vascular calcification and diabetes.

Methods Popliteal arteries, harvested from diabetic patients undergoing limb amputation, and non-atherosclerotic internal thoracic arteries, were stained for a range of DNA damage markers. Human coronary artery smooth muscle cells (vSMCs) were used for an in vitro calcification model (5mM βGP and 2.6mM CaCl2) in the presence or absence of a diabetic environment (high glucose (25mM), or control (5mM) DMEM respectively. DNA damage was induced using 200μM H2O2 and SIRT1 was activated or inhibited pharmacologically using SRT1720 or Sirtinol respectively. DNA damage was assessed via a comet assay, γH2A.X and p21 immunocytochemistry, whilst DNA damage-induced SIRT1 activation was assessed via nuclear fractionation and western blotting. Activation of the MRN DNA damage response pathway was confirmed by chIP qPCR and western blot analysis.

Results Alizarin red, Alkaline phosphatase activity and RUNX2 and OCN qPCR confirmed development of calcification during H2O2 treatment, which was reduced with SIRT1 activation. Immunohistochemistry demonstrated enhanced DNA damage marker expression in diabetic vessels compared to non-diabetic control ITA tissue, while SMCs harvested from diabetic patients also showed a significant reduction in SIRT1 expression (p<0.01) and elevated p53 expression (p<0.01) compared to cells from non-diabetic patients. SIRT1 expression was reduced in cells following high glucose treatment, in conjunction with increased protein expression of the DNA damage marker, γH2A.X (p<0.05). The comet assay showed a significant increase in DNA damage in both osteogenic and high glucose conditions following H2O2 treatment, which also caused an increase in SIRT1 nuclear translocation. Activation of SIRT1 significantly reduced H2O2 induced DNA damage (p<0.01) and increased recovery after 3h (p<0.005). In addition, deacetylation of MRE11, RAD50 (p<0.01) and NBS1 (MRN) (p<0.005) correlated with SIRT1 activation, and an increase in their phosphorylation following H2O2 treatment.

Conclusions This study demonstrates that SIRT1 protects against H2O2 induced DNA damage and subsequent calcification within a diabetic environment via activation of the MRN complex. The loss of SIRT1 within the calcified vessels of diabetic patients may contribute to a defective DNA repair mechanism, caused by the absence of SIRT1, resulting in a reduction of MRN deacetylation and thus activation of the DNA damage response, and could be an appropriate target for potential therapeutic intervention for vascular calcification.

Conflict of interest N/A

  • SIRT1
  • DNA Damage
  • Diabetes

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