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Pietro Maria Bertelli, Elisa Peixoto, Christina O'Neill, Jasenka Guduric-Fuchs, Lynsey-Dawn Allen, Alan W Stitt, Reinhold Medina; Endothelial Cellular Senescence in the Pathogenesis of Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3554.
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© ARVO (1962-2015); The Authors (2016-present)
Diabetic Retinopathy (DR) is the most common microvascular complication in diabetic patients. Diabetes is associated to endothelial dysfunction, which is characterised by impairment in vascular tone, vascular permeability, increase inflammation, deficit in blood perfusion and vessel loss. Moreover, links to ageing and cellular senescence have been suggested. In this study, we aim to evaluate the impact of the diabetic milieu on retinal endothelial cells in relation to senescence, to gain new insights into the biology of DR
Human Retinal Endothelial Cells (HRECs) were cultured in normal and high-glucose (NG: 5mM D-glucose; HG: 25mM D-Glucose). Population Doubling Level (PDL) was calculated and cellular senescence quantified with Senescence Associated (SA)-β-Galactosidase staining (Cell Signalling-9860). Cellular functionality was tested using a 3D tube formation assay (Corning Matrigel-356231). The Oxygen-Induced Retinopathy (OIR) mouse model was used to assess senescence in vivo. Control and OIR eyes were enucleated at P14 and retinas were dissected for mRNA (n=4) and flat mounting (n=3). Gene expression profile was assessed via RT-qPCR. ANOVA and two-tailed t-test were used for statistical analysis
Cumulative PDLs were significantly lower in HG-HRECs when compared to NG-HRECs (p<0.001), from 30 days until cells reached their Hayflick limit. The number of SA-β-Galactosidase positive cells was significantly higher in late-passage HG-HRECs when compared to matched late-passage NG-HRECs (p<0.001). Tube formation assay indicated that 4-week HG treated cells were less tubulogenic than NG-HRECs (p<0.05). In addition, these HG treated HRECs exhibited significant upregulation of IL8 (p<0.001), a major SASP component. Analysis of OIR mouse retinas by SA-β-Galactosidase staining showed higher senescence in ischaemic retinas, predominantly in the central retina (p<0.05). This was in agreement with an increased gene expression of senescent markers, such as TGFβ1 and IGFBPs by RT-qPCR (p<0.001)
Our results indicate that diabetic conditions induce cellular senescence in HRECs in vitro, which is coupled with functional impairment. Furthermore, we provide evidence to demonstrate that there is accumulation of senescent cells in the ischaemic mouse retina
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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