July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
RUNX1 Regulation of Epithelial-Mesenchymal Transition in Proliferative Vitreoretinopathy
Author Affiliations & Notes
  • Dhanesh Amarnani
    Ophthalmology, Schepens Eye Research Institute/ Mass Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Leslie Ramos
    Ophthalmology, Schepens Eye Research Institute/ Mass Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Santiago Delgado-Tirado
    Ophthalmology, Schepens Eye Research Institute/ Mass Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Whitney Greene
    US Army of Surgical Institute, Texas, United States
  • Heuy-Ching Hetty Wang
    US Army of Surgical Institute, Texas, United States
  • Leo A. Kim
    Ophthalmology, Schepens Eye Research Institute/ Mass Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Joseph Arboleda-Velasquez
    Ophthalmology, Schepens Eye Research Institute/ Mass Eye and Ear Infirmary, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Dhanesh Amarnani, None; Leslie Ramos, None; Santiago Delgado-Tirado, None; Whitney Greene, None; Heuy-Ching Wang, None; Leo Kim, None; Joseph Arboleda-Velasquez, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5811. doi:https://doi.org/
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      Dhanesh Amarnani, Leslie Ramos, Santiago Delgado-Tirado, Whitney Greene, Heuy-Ching Hetty Wang, Leo A. Kim, Joseph Arboleda-Velasquez; RUNX1 Regulation of Epithelial-Mesenchymal Transition in Proliferative Vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5811. doi: https://doi.org/.

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

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Abstract

Purpose : Proliferative Vitreoretinopathy (PVR) is a complication after ocular trauma characterized by the formation of retinal membranes. PVR is thought to be mediated via epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells. RUNX transcription factors play a key role in biological processes involving EMT in developmental and cancer biology. We hypothesize that RUNX1 mediates EMT in PVR

Methods : Immunofluorescence staining (IF) was performed on patient-derived PVR membranes (n=3) for RUNX1. EMT was induced in ARPE-19 and primary cultures from patient-derived PVR membranes (C-PVR) by 10ng/ml TGF-β2, TNF-α, IL-6 and a combination treatment of all three growth factors. Expression of EMT markers Occludin and N-Cadherin were evaluated by qRT-PCR and Western blot analyses 3 days post treatment in both cell types. RUNX1 expression was examined via qRT-PCR and Western blot analyses. RUNX1 activity was inhibited by Ro5-3335 and expression of α-SMA was evaluated by IF. RUNX1 knockdown was performed using siRNA and levels of EMT markers were evaluated 2 days post treatment in both cell types. RUNX1 inhibition using Ro5-3335 was tested in an in vitro wound-healing assay using retinal pigment epithelial cells derived from induced pluripotent stem cells (iPS-RPE).

Results : RUNX1 was detected in a subpopulation of cells in PVR membranes. In ARPE-19 cells, TGF-β2, TNF-α and combination treated cells led to a decrease in mRNA levels of Occludin (2-fold) and an increase in mRNA (1.5-fold) and protein of N-Cadherin. In C-PVR cells, TGF-β2 and combination treated cells led to a reduction in mRNA levels of Occludin (2-fold) and an increase in mRNA levels (2-fold) and protein of N-Cadherin. An increase in RUNX1 expression (P < 0.05) was observed in both cell types. In ARPE-19 cells, EMT and fibrosis were inhibited by Ro5-3335 and confirmed by diminished α-SMA staining. RUNX1 knockdown partially inhibited EMT as assessed by a no significant change in E-Cadherin and N-Cadherin mRNA and protein levels. In iPS-derived RPE, Ro5-3335 (100uM) decreased vitreous treatment-induced α-SMA staining 14 days post treatment.

Conclusions : Our results support our hypothesis of a critical role for RUNX1 as a mediator of EMT and fibrosis in PVR across multiple cell models: ARPE-19, C-PVR, and iPS-RPE cells. Modulation of RUNX1 can potentially be a method for partial or complete inhibition of EMT preventing the progression of PVR.

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

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