June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Regulation of miR-21 expression and exosomes-mediated secretion in human retinal pigmented epithelial cells (HuRPE) exposed to hypoxia
Author Affiliations & Notes
  • Menaka Thounaojam
    Ophthalmology, Augusta University, Augusta, Georgia, United States
  • Diana Gutsaeva
    Ophthalmology, Augusta University, Augusta, Georgia, United States
  • Pamela M Martin
    Biochemistry and Molecular Biology , Augusta University, Augusta, Georgia, United States
  • Manuela Bartoli
    Ophthalmology, Augusta University, Augusta, Georgia, United States
  • Footnotes
    Commercial Relationships   Menaka Thounaojam, None; Diana Gutsaeva, None; Pamela Martin, None; Manuela Bartoli, None
  • Footnotes
    Support  EY022416
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1047. doi:
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      Menaka Thounaojam, Diana Gutsaeva, Pamela M Martin, Manuela Bartoli; Regulation of miR-21 expression and exosomes-mediated secretion in human retinal pigmented epithelial cells (HuRPE) exposed to hypoxia. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1047.

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

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Abstract

Purpose : Expression and regulation of microRNAs (miRs) in the retinal tissue is still under investigation. We have identified the retinal pigmented epithelium as an important source of several miRs and as an active producer of the miRs “cargos” exosomes. Here we have studied the mechanisms of production and exosome-mediated release of miR-21, a non-coding small RNA that we have previously studied in the context of its activity in promoting retinal neovascularization and vascular inflammation.

Methods : Primary cultures of human retinal pigmented epithelial cells (HuRPE) and ARPE18 were cultured according to their manufacturer’s recommendation. The cells were let polarize for 7-10 days before exposure to hypoxic conditions (pO2= 2+/-0.5) for different times (6, 12 and 24 hours). Exosomes were isolated from the cultured medium by following serial high speed centrifugation. Exosomes yield and identity was confirmed by TEM and gold immunostaining for the specific markers CD65 and flotilin. QPCR was performed to assess miR-21 expression pattern. Angiogenic assays (tube formation, up-regulation of collagenases –MMP2, MMP9) were performed on human retinal endothelial cells (HuREC) exposed to different dilution of isolated exosomes.

Results : Hypoxia significantly increased HuRPE and ARPE18 production of miR-21 and exosomes formation. MiR-21 levels were significantly elevated in exosomes secreted by HuRPE and ARPE18 exposed to hypoxia (peak at 6-12 hours). Exosomes isolated from hypoxic HuRPE and ARPE18 conditioned medium stimulated angiogenic features such as tube formation and MMPs activation in HuREC with highest activity measured at 6 and 12 hours. Same effects were produced by transfecting the cells with miR-21 mimic. Blockade of miR-21 biological effects in cells by transfecting cells with a specific miR-21 inhibitor (a.miR-21), significantly reduced the hypoxic exosomes pro-angiogenic effects.

Conclusions : Our data demonstrate production and exosomes-mediated release of miRs, i.e. miR-21, in HuRPE subjected to pathologically relevant simulants (such as hypoxia/ischemia), thus suggesting a potential role for this mechanism in promoting epigenetic changes as well as fine-tuned regulated gene expression in the diseased retina.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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