July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Ceramide-Induced Mitochondrial Changes in Retinal Endothelial Cells
Author Affiliations & Notes
  • Yan Levitsky
    Chemistry, Physiology, Michigan State University, East Lansing, Michigan, United States
    DO/PhD Physician Scientist Training Program, Michigan State University, East Lansing, Michigan, United States
  • Sandra S Hammer
    Physiology, Michigan State University, East Lansing, Michigan, United States
  • Todd Lydic
    Molecular Metabolism and Disease - Mass Spectrometry Core, Michigan State University, East Lansing, Michigan, United States
    Physiology, Michigan State University, East Lansing, Michigan, United States
  • David Pegouske
    Chemistry, Michigan State University, East Lansing, Michigan, United States
  • Kiera Fisher
    Physiology, Michigan State University, East Lansing, Michigan, United States
  • Denis Proshlyakov
    Chemistry, Michigan State University, East Lansing, Michigan, United States
  • Julia V Busik
    Physiology, Michigan State University, East Lansing, Michigan, United States
  • Footnotes
    Commercial Relationships   Yan Levitsky, None; Sandra Hammer, None; Todd Lydic, None; David Pegouske, None; Kiera Fisher, None; Denis Proshlyakov, None; Julia Busik, None
  • Footnotes
    Support  1R01EY028049
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2707. doi:https://doi.org/
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      Yan Levitsky, Sandra S Hammer, Todd Lydic, David Pegouske, Kiera Fisher, Denis Proshlyakov, Julia V Busik; Ceramide-Induced Mitochondrial Changes in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2707. doi: https://doi.org/.

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

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Abstract

Purpose : Diabetic retinopathy (DR) is a sight threatening complication of diabetes. Pathogenic mechanisms involve effects of hyperglycemia, dyslipidemia, and chronic inflammation causing break down of the blood retinal barrier and DR progression. Acid sphingomyelinase (ASMase) induced ceramide accretion is a potent mediator of retinal endothelial cell (REC) apoptosis but detailed mechanistic insights are lacking. This study aims to elucidate the role of mitochondrial ceramide accretion in REC apoptosis.

Methods : Retinal mitochondria were isolated from streptozotocin-induced diabetic and control rats. Human and bovine REC were cultured by conventional techniques and on microrespirometer chips. Mitochondria were prepared by differential centrifugation and a magnetically-activated cell sorting (MACS) kit. Purity was assessed by immunoblotting for mitochondrial, lysosomal, peroxisomal and cell membrane marker proteins. Sphingolipid changes were assessed by mass spectrometry. Mitochondrial function was assessed by microrespirometry.

Results : Mitochondria prepared by differential centrifugation showed presence of lysosomal membranes which were undetectable after MACS purification. Retinal mitochondria obtained from 7-week diabetic rats revealed a 1.6-fold decrease in sphingomyelin to ceramide ratio compared to control (n=3; p<0.05). Retinal mitochondria from 36-week diabetic rats (n=4) showed a 1.4-fold increase in short chain ceramides compared to control (n=3; p<0.01). Bio-mimetic microrespirometric assessment revealed no loss of BREC respiratory activity during six hours of continuous media flow with intermittent pauses for activity assessment. Pre-treatment of BREC with an ASMase inhibitor revealed a 1.75-fold increase in maximal respiratory capacity compared to control cells. Changes to basal respiration were not observed when ASMase inhibitor was added to media during respiratory assessment.

Conclusions : MACS mitochondrial isolation results in pure preparations amenable to “-omic” analyses. Short- and long-term diabetes results in increased mitochondrial ceramide and decreased mitochondrial sphingomyelin consistent with ASMase dependent mCer accretion. Inhibition of ASMase in REC results in increased maximal, but not basal, respiratory rates. These results support the hypothesis that ASMase-dependent mitochondrial ceramide accumulation leads to inhibition of oxidative phosphorylation and REC cell damage in diabetic environment.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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