May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Mechanism of Pericyte Apoptosis Following Glucose Exposure
Author Affiliations & Notes
  • J.G. Browne
    Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland
  • R. Kane
    Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland
  • C. Godson
    Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland
  • T.G. Cotter
    Tumor Biology Laboratory, Biochemistry Department,, University College Cork,, Lee Maltings, Prospect Row, Cork,, Ireland
  • C.J. O'Brien
    Institute of Ophthalmology, Mater Miseriocdiae Hospital, Dublin, Ireland
  • Footnotes
    Commercial Relationships  J.G. Browne, None; R. Kane, None; C. Godson, None; T.G. Cotter, None; C.J. O'Brien, None.
  • Footnotes
    Support  HRB Grant NS19/2002
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3227. doi:
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      J.G. Browne, R. Kane, C. Godson, T.G. Cotter, C.J. O'Brien; Mechanism of Pericyte Apoptosis Following Glucose Exposure . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3227.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose:Diabetic retinopathy (DR) is among the most common diabetic complications and one of the leading causes of vision loss in the working age population. Retinal changes characteristic of the onset of DR include microaneurysms, haemorrhages and the formation of some areas of accelularity. One of the earliest changes observed in retinal microvessels is the selective loss of pericytes. It has been shown that caspase–8–mediated cleavage of Bid to its active form tBid followed by its redistribution to the mitochondria where it targets the outer mitochondraial membrane and induces conformational changes in Bak and Bax and, in so doing triggers cytochrome c release into the cytosol. Apoptosome formation can then take place resulting in activation of caspase–3 and other effector caspases, ultimately causing apoptotic cell death. Therefore we decided to investigate this mechanism in relation to pericyte apoptotis. Methods:Human retinal pericytes were cultured in MCDB 131, 10% FBS, 2mM L–glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin. On becoming confluent these cells were serum starved (0.5% FBS) for 24 hrs. These cells were then cultured in 5mM to 40mM glucose in increments of 5mM for 24 hrs in media containing 0.5% FBS. Total RNA was extracted using Tri–ReagentTM and reverse–transcribed using standard protocols. The cDNA obtained was used for PCR and TaqmanTM real–time PCR analysis of Bax, Bak and Bid expression. Western blotting was also used to detect the presence of Bid and caspase–8 and their active truncated forms. Results:Increased levels of Bax, Bak and Bid mRNA expression with maximal expression at 30mM glucose, while changes in levels of Bid, tBid, caspase–8 and cleaved caspase–8 fragments were detected using western blotting. The appearance of a low molecular weight DNA ladder was used as a marker of apoptosis. This DNA laddering was observed in parallel with caspase–8 activation. Conclusions:These results demonstrate that in high glucose conditions pro–apoptotic genes such as Bax, Bak and Bid are upregulated in retinal pericytes within a time period of 24 hrs. Active caspase–8 levels also increase as glucose concentration increases. These findings indicate a possible mechanism for glucose induced cell death in human retinal pericytes.

Keywords: gene/expression • diabetic retinopathy • apoptosis/cell death 
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