July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Extracellular and intracellular miR-21 promotes oxidative stress and inflammation in the diabetic retinal microvasculature by activation of TLR-7
Author Affiliations & Notes
  • Manuela Bartoli
    Ophthalmology, Augusta University, Augusta, Georgia, United States
  • Menaka Thounaojam
    Ophthalmology, Augusta University, Augusta, Georgia, United States
  • Folami Lamoke Powell
    Biochemistry and Molecular Biology, Augusta University, Augusta, Georgia, United States
  • Ravirajsinh Jadeja
    Biochemistry and Molecular Biology, Augusta University, Augusta, Georgia, United States
  • Pamela M Martin
    Biochemistry and Molecular Biology, Augusta University, Augusta, Georgia, United States
  • Diana Gutsaeva
    Ophthalmology, Augusta University, Augusta, Georgia, United States
  • Footnotes
    Commercial Relationships   Manuela Bartoli, None; Menaka Thounaojam, None; Folami Powell, None; Ravirajsinh Jadeja, None; Pamela Martin, None; Diana Gutsaeva, None
  • Footnotes
    Support  EY022146
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3003. doi:
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      Manuela Bartoli, Menaka Thounaojam, Folami Lamoke Powell, Ravirajsinh Jadeja, Pamela M Martin, Diana Gutsaeva; Extracellular and intracellular miR-21 promotes oxidative stress and inflammation in the diabetic retinal microvasculature by activation of TLR-7. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3003.

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

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Abstract

Purpose : We have previously shown that activation of toll-like receptor 4 and 7 in human retinal endothelial cells (HuREC) promotes xanthine oxidase (XOD) expression in the diabetic retinal microvasculature. In turn increased XOD activity leads to oxidative stress and inflammation, thus contributing to diabetic retinopathy (DR). Here we have performed in vitro and in vivo studies to investigate the specific contribution of miR-21 to this process.

Methods : In situ hybridation and qPCR were performed to detect miR-21 expression and distribution in retinas of streptozotocin-induced diabetic rats (STZ-rats) after 8 weeks of hyperglycemia. Human retinal endothelial and epithelial cells (HuREC and HuRPE, respectively) were cultured in 5mM glucose (NG)-containing medium and switched to 25mM (LG) glucose for 48hours to mimic glucidic stress. L-glucose (25mM - LG), was used as osmotic control. Exosomes (miR-cargoes, EV) were isolated by differential centrifugation methods and identified/selected by immunoblotting of specific markers. TLR-7 expression was analyzed by qPCR and immunoblotting. HuREC and HuRPE were transfected with 50nM miR-21 mimic or the miR-21 inhibitor, antagomiR-21. Non-targeted miR and antagomiR were used as control. Cell ROX was used to measure production of reactive oxygen species (ROS).

Results : MiR-21 expression was increased in retinas of STZ-rats at 8 weeks of hyperglycemia. In situ hybridization showed miR-21 production in retinal microvasculature and, mostly, in retinal pigmented epithelium (RPE). XOD showed similar expression pattern and distribution. EVs isolated from HG-treated HuREC and HuRPE presented increased levels of miR-21 compared to EVs isolated by cells exposed to NG or LG. Interestingly, HuRPE showed much higher levels of EVs and miR-21 than HuREC. TLR7 and XOD expression were up-regulated in HuREC exposed to HG and antagomiR-21 blocked this effect. Furthermore, EVs isolated from HuREC and HuRPE exposed to HG promoted TLR7 and XOD expression and ROS formation in HuREC cultured in NG. This effect was blocked by transfection of the cells with antagomiR-21.

Conclusions : Our studies demonstrate the existence of novel mechanism of oxidative and inflammatory responses in the diabetic retinal microvasculature involving miR-21 autocrine and paracrine interaction with the TLR-7-XOD pathway.

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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