June 2020
Volume 61, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2020
Metabolic alterations during TGFβ2-induced EMT in retinal pigment epithelial (RPE) cells
Author Affiliations & Notes
  • Daisy Yao Shu
    Schepens Eye Research Institute of Mass Eye and Ear, Boston, Massachusetts, United States
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
  • Erik Butcher
    Schepens Eye Research Institute of Mass Eye and Ear, Boston, Massachusetts, United States
    Harvard University, Cambridge, Massachusetts, United States
  • Siwei Cai
    Schepens Eye Research Institute of Mass Eye and Ear, Boston, Massachusetts, United States
  • Ilakya Senthilkumar
    Schepens Eye Research Institute of Mass Eye and Ear, Boston, Massachusetts, United States
    Northeastern University, Boston, Massachusetts, United States
  • Scott Frank
    Schepens Eye Research Institute of Mass Eye and Ear, Boston, Massachusetts, United States
    Boston College, Chestnut Hill, Massachusetts, United States
  • Kiran Kurmi
    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States
  • Magali Saint-Geniez
    Schepens Eye Research Institute of Mass Eye and Ear, Boston, Massachusetts, United States
    Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Daisy Shu, None; Erik Butcher, None; Siwei Cai, None; Ilakya Senthilkumar, None; Scott Frank, None; Kiran Kurmi, None; Magali Saint-Geniez, None
  • Footnotes
    Support  Grimshaw-Gudewicz Charitable Foundation; National Council of Scientific and Technological Development—Brazil (PDE 210474/2014-9); The Iraty Award; NEI Core Grant P30EYE003790; Department of Defense, Spinal Vision Research Program under Award no. VR180132
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 3181. doi:
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    • Get Citation

      Daisy Yao Shu, Erik Butcher, Siwei Cai, Ilakya Senthilkumar, Scott Frank, Kiran Kurmi, Magali Saint-Geniez; Metabolic alterations during TGFβ2-induced EMT in retinal pigment epithelial (RPE) cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3181.

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

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Abstract

Purpose : Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE) plays a key role in fibrotic retinal diseases including proliferative vitreoretinopathy and subretinal fibrosis in age-related macular degeneration. We previously showed that metabolic dysfunction by repressing PGC-1α induced EMT in RPE. Here, we investigated the metabolic alterations in RPE caused by transforming growth factor-beta 2 (TGFβ2), a potent EMT inducer.

Methods : Matured ARPE-19 were treated with TGFβ2 (10 ng/ml) for up to 72h. Glycolysis and oxidative phosphorylation (OXPHOS) were examined by high-resolution respirometry. Gene expression of EMT and metabolic markers were assessed using qPCR. Vimentin expression was assessed with immunofluorescence. ARPE-19 were transfected with CellLight Mitochondria-GFP and imaged using confocal microscopy. Parameters of mitochondrial morphology were extracted using automated image segmentation. Citrate synthase activity was assessed using the MitoCheck assay. ATP content was measured using a bioluminescent assay. Cytoplasmic and secreted metabolites were measured by LC/MS metabolomics.

Results : TGFβ2 induced RPE to transdifferentiate into spindle-shaped mesenchymal cells with increased vimentin protein expression and EMT gene expression (Col1A1: 1.2 vs 6.8, p < 0.02; CTGF: 1.1 vs 3.4 p < 0.02). High-resolution respirometry revealed a reduction in maximum OXPHOS levels (OCR = 0.80 vs 0.41 pmol/min/μg, p < 0.0047) and increased glycolytic reserve (ECAR = 4.6 vs 17.9 μpH/mol/μg, p < 0.0119), consistent with reduced expression of OXPHOS genes (ATP5O: 1 vs 0.7, p < 0.005; NDUFB5: 1 vs 0.5, p < 0.0001) and increased glycolysis (PFKFB3: 1.2 vs 4.5, p < 0.004). TGFβ2 reduced citrate synthase activity (3.8 vs 3.0 pmol/ml/μg, p < 0.0067) and ATP content (1 vs 0.23, p < 0.0005). Metabolomic profiling revealed changes in glycolytic, OXPHOS and nucleotide synthesis pathways with TGFβ2. Importantly, TGFβ2 suppressed PGC-1α gene expression (1 vs 0.13, p < 0.0024) and disrupted mitochondrial network morphology.

Conclusions : TGFβ2-induced EMT of RPE is accompanied by a profound metabolic shift towards increased glycolysis and reduced OXPHOS. PGC-1α, a master regulator of mitochondrial biogenesis, may mediate TGFβ2-induced disruption of metabolic function. Targeting metabolic pathways is a promising therapeutic approach for treating fibrotic retinal diseases.

This is a 2020 ARVO Annual Meeting abstract.

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