June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
miR-15a/16 inhibits TGF-beta3 and SMAD2/3 signaling and maintains retinal endothelial barrier
Author Affiliations & Notes
  • Eun-Ah Ye
    Anatomy and Cell biology, Wayne State University, Detroit, Michigan, United States
  • Jena J Steinle
    Anatomy and Cell biology, Wayne State University, Detroit, Michigan, United States
  • Footnotes
    Commercial Relationships   Eun-Ah Ye, None; Jena Steinle, None
  • Footnotes
    Support  RO1EY022045
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2515. doi:
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      Eun-Ah Ye, Jena J Steinle; miR-15a/16 inhibits TGF-beta3 and SMAD2/3 signaling and maintains retinal endothelial barrier
      . Invest. Ophthalmol. Vis. Sci. 2017;58(8):2515.

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

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Abstract

Purpose : A key risk factor of diabetic retinopathy is hyperglycemia, and molecular mechanisms underlying hyperglycemia and diabetic retinopathy in association with microRNA are still poorly understood. There are increasing amounts of evidence on microRNA as a mediator of pathological mechanisms in diabetic retinopathy. The purpose of this study was to test the hypothesis that miR-15a/16 inhibit TGF-beta3 and SMAD2/3 signaling, thus maintaining retinal endothelial barrier.

Methods : Human primary retinal endothelial cells (REC) were maintained in normal (5mM) glucose or transferred to high glucose medium (25 mM) for 3 days. REC were transfected with miRNA mimics (hsa-miR-15a-5p and -16-5p, at a final concentration of 30 nM) 48 hours before cell harvest. Western blot analyses were performed to measure the levels of proteins of interest. A permeability assay was performed to measure solute flux through the monolayer of cultured REC. In addition, retinal lysates of miR-15a-transgenic mice were analyzed for the changes of protein levels.

Results : We demonstrated that overexpression of miR-15a/16 resulted in decreased TGF-beta3 signaling and VEGF levels in REC cultured under high glucose conditions. In addition, levels of the tight junction proteins ZO-1 and occludin were elevated in REC overexpressing miR-15a and -16. Overexpression of miR-15a in REC played a role in reducing cellular permeability, potentially through inhibition of TGF beta3 signaling in high glucose conditions. Using miR-15a-transgenic mice, we demonstrated the regulatory role of miR-15a on TGF-beta signaling and key tight junction proteins in vivo.

Conclusions : miR-15a/16 played a role in 1) inhibiting TGF beta3, SMAD2/3, and VEGF signaling and 2) increasing levels of ZO-1 and occludin in REC cultured in high glucose conditions. Additionally, miR-15a overexpression resulted in decreased levels of endothelial permeability in vitro. Our in vivo data confirmed the regulatory roles of miR-15a on TGF beta3 signaling and the tight junction proteins in the retina. Therefore, we suggest that miR-15a/16 maintains retinal endothelial barrier by reducing TGF-beta3 signaling and increasing the levels of tight junction proteins.

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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