July 2018
Volume 59, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2018
MicroRNAs as mediators of fibrosis in an in vitro model of proliferative vitreoretinopathy
Author Affiliations & Notes
  • Patricia Sanchez-Diaz
    Rosenberg School of Optometry, University of the Incarnate Word, San Antonio, Texas, United States
  • Whitney Greene
    Ocular Trauma, United States Army Institute of Surgical Research, San Antonio, Texas, United States
  • Patrick A Clark
    Rosenberg School of Optometry, University of the Incarnate Word, San Antonio, Texas, United States
  • Teresa A Burke
    Ocular Trauma, United States Army Institute of Surgical Research, San Antonio, Texas, United States
  • Heuy-Ching Hetty Wang
    Ocular Trauma, United States Army Institute of Surgical Research, San Antonio, Texas, United States
  • Footnotes
    Commercial Relationships   Patricia Sanchez-Diaz, None; Whitney Greene, None; Patrick Clark, None; Teresa Burke, None; Heuy-Ching Wang, None
  • Footnotes
    Support  U.S. Army Clinical Rehabilitative Medicine Research Program (CRMRP) and Military Operational Medicine Research Program (MOMRP). WG was supported by the National Research Council. PCSD and PAC were supported by Intramural Research Funds from the University of the Incarnate Word.
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 5388. doi:
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    • Get Citation

      Patricia Sanchez-Diaz, Whitney Greene, Patrick A Clark, Teresa A Burke, Heuy-Ching Hetty Wang; MicroRNAs as mediators of fibrosis in an in vitro model of proliferative vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5388.

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

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Abstract

Purpose : This study aims to unravel the molecular mechanisms involved in the phenotypic changes that occur in RPE cells after penetrating ocular trauma. We hypothesized that vitreous induces fibrotic changes in wounded RPE cells that will be mediated, at least in part, by microRNA changes.

Methods : We used retinal pigment epithelial cells derived from induced pluripotent stem cells (iPS- RPE cells) as an in vitro model for PVR. iPS-RPE cells were plated onto matrigel-coated transwells and cultured for 60 days until fully confluent and pigmented. Cell monolayers were then scratched to create a wound. To recapitulate the in vivo conditions that lead to PVR, cells were treated with 5% fresh vitreous immediately after wounding. Unscratched cells without vitreous were included as controls.
Gene expression of 84 pro-fibrotic miRNAs was measured by qPCR in wounded iPS-RPE cells on days 3 and 12 post-scratch. A 2-fold change in the miRNA levels compared to controls was used as threshold to identify differentially expressed miRNAs (DEmiRs).
An alpha smooth muscle actin (α-SMA) reporter assay was used to screen for potential miRNAs involved in vitreous-induced RPE fibrosis. ARPE19 retinal epithelial cells expressing the luciferase gene under the control of the α-SMA promoter were transfected with miRNA mimics and inhibitors of the DEmiRs and the activity of the α-SMA promoter measured using a luminescence assay.

Results : Focused PCR array analysis revealed a distinctive pattern of pro-fibrotic miRNA expression during wound healing in our in vitro model of PVR. Compared to unscratched cells without vitreous, differential expression of 11 fibrosis-associated miRNAs (>5-fold change, P<0.05) was found in scratched iPS-RPE cells incubated with 5% vitreous. Predicted targets of these DEmiRs (TargetScan) included genes involved in fibrosis, angiogenesis, inflammation, epithelial mesenchymal transition, and tissue remodeling. The luciferase reporter assay showed increased α-SMA promoter activity in ARPE19 cells after transfection with miRNA mimics (compared to scramble microRNA).

Conclusions : The DEmiRs identified in our in vitro model of PVR might be involved in RPE transdifferentiation into myofibroblast-like cells and thus be a marker and a prospective therapeutic target for PVR, a leading cause of vision loss in children and young adults for which there is not a successful treatment.

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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