Conference Abstracts

Abstracts for conferences I’ve presented at:

 


March 12th 2020

Shu DY, Butcher E, Cai S, Senthilkumar I, Frank S, Saint-Geniez M. Metabolic reprogramming in TGFβ2-induced EMT of the retinal pigment epithelium [Abstract]. 3rd Annual MEE Joint Research Symposium, Starr Centre (Boston, MA) (Link).

Metabolic reprogramming in TGFβ2-induced EMT of the retinal pigment epithelium

Daisy Y. Shu1,2, Erik Butcher1,3, Siwei Cai1, Ilakya Senthilkumar1, Scott Frank1, Magali Saint-Geniez1,2

1Schepens Eye Research Institute of Mass Eye and Ear, 2Harvard Medical School, Department of Ophthalmology, 3Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE) is a critical process in fibrotic retinal diseases including proliferative vitreoretinopathy and subretinal fibrosis in age-related macular degeneration. We investigated the metabolic alterations in RPE caused by transforming growth factor-beta 2 (TGFβ2), a potent EMT inducer. Matured ARPE-19 were treated with TGFβ2 (10 ng/ml) for up to 72h. TGFβ2 induced RPE to transdifferentiate into spindle-shaped mesenchymal cells with increased vimentin expression on immunofluorescence confocal microscopy and increased EMT gene expression (α-SMA, Snai1/2, Col1A1, fibronectin, CTGF, MMP2) using RT-qPCR. High-resolution respirometry (Seahorse XF24 Extracellular Flux Analyzer) showed that TGFβ2 reduced maximum OXPHOS levels and increased glycolytic reserve, consistent with reduced expression of OXPHOS genes (ATP5O, COX4I1, COX5B, NDUFB5) and increased glycolysis genes (PFKFB3, PGK1). TGFβ2 also reduced citrate synthase activity and ATP content as assessed using the MitoCheck Assay and Sigma Bioluminescent Assay Kit, respectively. Importantly, TGFβ2 suppressed PGC-1α gene expression, a master regulator of mitochondrial biogenesis, suggesting that EMT is accompanied by mitochondrial dysfunction. Taken together, our data shows a profound metabolic shift towards increased glycolysis and reduced OXPHOS in TGFβ2-induced EMT of RPE. Targeting metabolic pathways may be a promising therapeutic approach for treating fibrotic retinal diseases.


May 5th 2020

Shu DY, Butcher E, Cai S, Senthilkumar I, Frank S, Kurmi K, Saint-Geniez M. Metabolic alterations during TGFβ2-induced EMT in retinal pigment epithelial (RPE) cells. [Abstract]. ARVO Annual Meeting, May 2020 (Baltimore, MD)

Metabolic alterations during TGFβ2-induced EMT in retinal pigment epithelial (RPE) cells

Daisy Y. Shu1, Erik Butcher1, Siwei Cai1, Ilakya Senthilkumar1, Scott Frank1, Kiran Kurmi2, Magali Saint-Geniez1

1Schepens Eye Research Institute, Mass Eye and Ear, Harvard Medical School, 2Department of Cell Biology, Harvard Medical School, Boston, MA 02115

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.


April 7th 2020

Shu DY, Butcher E, Cai S, Senthilkumar I, Frank S, Saint-Geniez M. Paradoxical Effect of TNFα on Mitochondrial Function and Metabolic Activity in the Retinal Pigment Epithelium (RPE) [Abstract]. Experimental Biology, American Society for Pathology (San Diego, CA) (Link).

Paradoxical Effect of TNFα on Mitochondrial Function and Metabolic Activity in the Retinal Pigment Epithelium (RPE)

Daisy Y. Shu, Erik Butcher, Siwei Cai, Ilakya Senthilkumar, Scott Frank, Magali Saint-Geniez

Schepens Eye Research Institute, Mass Eye and Ear, Harvard Medical School

The retinal pigment epithelium (RPE) acts as a metabolic gatekeeper between photoreceptors and the choroidal vasculature to maintain healthy retinal function. RPE dysfunction is a key feature of age-related macular degeneration (AMD), the leading cause of blindness in developed countries. Tumor necrosis factor-alpha (TNFα), a potent pro-inflammatory cytokine, has been implicated in the pathogenesis of AMD. Growing evidence supports metabolic dysfunction as another key mechanism driving AMD. To date, there is no literature on the metabolic effects of TNFα on RPE and thus, this study sheds light on the impact of TNFα on mitochondrial morphology and metabolic function in RPE. Matured ARPE-19 (human RPE cell line) were treated with TNFα (10 ng/ml) for up to 72h. TNFα induced ARPE-19 to elongate into spindle-shaped cells, reminiscent of epithelial-mesenchymal transition (EMT). However, qPCR showed that TNFα reduced the expression of EMT genes (α-SMA, Col1A1, fibronectin, MMP2, CTGF) indicating that the elongated cells were not mesenchymal in nature. To explore the effects of TNFα on metabolism, two major energy-generating pathways, oxidative phosphorylation (OXPHOS) and glycolysis were assessed by Seahorse high-resolution respirometry and qPCR. The Seahorse Mito Stress Test revealed an increase in basal respiration but reduced spare respiratory capacity following TNFα treatment. TNFα increased glycolytic reserve and capacity. Despite the increased OXPHOS and glycolysis, qPCR showed a significant reduction in expression of OXPHOS (ATP5O, COX4I1, COX5B, NDUFB5) and glycolysis (G6P, PFK1, PFKFB3, PGK1, LDHA) genes with TNFα treatment. ARPE-19 were transfected with mitochondria-tagged GFP and imaged using confocal microscopy. Parameters on mitochondrial morphology were extracted from the confocal images using automated image segmentation and revealed that TNFα disrupted mitochondrial network integrity. This was supported by qPCR data showing significantly reduced expression of genes regulating mitochondrial function (PGC-1α, TFAM, POLG, MFN1, MFN2, FIS1, OPA1). TNFα significantly elevated SOD2, a mitochondrial antioxidant enzyme, but not SOD1. Taken together, we find that TNFα robustly disrupts mitochondrial function and morphology in RPE, although shifting the bioenergetic profile in a paradoxical manner, i.e. TNFα raised the levels of basal respiration and glycolysis despite the suppression of genes regulating OXPHOS and glycolysis. These findings highlight the potential of targeting metabolic pathways in RPE as a promising therapeutic avenue for AMD. Further research is required to elucidate the mechanisms underlying these intriguing TNFα-driven metabolic changes.


April 21st 2020

Shu DY, Butcher E, Cai S, Senthilkumar I, Frank S, Saint-Geniez M.Mitochondrial dysfunction and metabolic reprogramming in retinal epithelial-mesenchymal transition [Abstract]. New York Academy of Sciences Mitochondria in Complex Diseases (New York, New York) (Link).

Mitochondrial dysfunction and metabolic reprogramming in retinal epithelial-mesenchymal transition

Daisy Y. Shu1,2, Erik Butcher1,3, Siwei Cai1, Ilakya Senthilkumar1, Scott Frank1, Magali Saint-Geniez, PhD1,2

1Schepens Eye Research Institute of Mass Eye and Ear, 2Harvard Medical School, Department of Ophthalmology, 3Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University

Epithelial-mesenchymal transition (EMT) is a key process in wound healing and fibrosis whereby epithelial cells lose their cellular adhesions and polarity, transforming into motile, extracellular matrix-producing mesenchymal cells. Transforming growth factor-beta (TGFβ) induces EMT in various cells, including the retinal pigment epithelium (RPE). TGFβ-induced EMT of RPE is critical in the pathogenesis of fibrotic retinal diseases leading to irreversible blindness. We investigated the metabolic changes associated with EMT in RPE. The human RPE cell line, ARPE-19, was treated with TGFβ2 (10 ng/ml) for up to 72h. TGFβ2 induced RPE to transdifferentiate into spindle-shaped mesenchymal cells with increased vimentin expression on immunofluorescence and increased EMT gene expression (α-SMA, Snai1/2, Col1A1, FN) using qPCR. High-resolution respirometry (Seahorse XF24) showed that TGFβ2 reduced OXPHOS levels and increased glycolytic reserve, consistent with reduced OXPHOS (ATP5O, COX4I1, COX5B) and increased glycolysis (PFKFB3) gene expression. TGFβ2 reduced citrate synthase activity and ATP content using the MitoCheck Assay and Sigma Bioluminescent Assay Kit, respectively. Staining with MitoTracker Orange revealed defects in mitochondrial morphology with TGFβ2, showing mitochondrial network loss with greater sphericity and fragmentation. Taken together, EMT is accompanied by mitochondrial dysfunction and a profound metabolic shift towards increased glycolysis and reduced OXPHOS.


November 9-13th 2020

Shu DY, Butcher E, Cai S, Senthilkumar I, Frank S, Saint-Geniez M. [Abstract]. Metabolic Rewiring and Mitochondrial Dysfunction in Transforming Growth Factor-Beta 2-Induced Retinal Epithelial-Mesenchymal Transition. American Society for Investigative Pathology (ASIP) PISA 2020 Conference (Link).

Metabolic Rewiring and Mitochondrial Dysfunction in Transforming Growth Factor-Beta 2-Induced Retinal Epithelial-Mesenchymal Transition

Daisy Y. Shu1,2, Erik Butcher1,3, Siwei Cai1, Ilakya Senthilkumar1, Scott Frank1, Magali Saint-Geniez, PhD1,2

1Schepens Eye Research Institute of Mass Eye and Ear 2Harvard Medical School, Department of Ophthalmology 3Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University

Introduction

Transforming growth factor-beta 2 (TGFβ2) is a key orchestrator of retinal wound healing through induction of epithelial-mesenchymal-transition (EMT) in retinal pigment epithelial cells (RPE), a pathogenic process driving fibrotic retinal diseases. We describe a previously unrecognized function of TGFβ2 in modulating mitochondrial morphology and metabolic function in human RPE cells.

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 (Seahorse XF24). Gene expression of metabolic and oxidative stress markers were assessed using qPCR. Mitochondrial morphology was assessed by confocal microscopy imaging of MitoTracker Orange-stained ARPE-19 and subsequent computational analysis of mitochondrial network parameters using ImageJ. Citrate synthase activity was assessed using the MitoCheck assay. ATP content was measured using a bioluminescent assay. Mitochondrial reactive oxygen species (ROS) and total cellular ROS was assessed using MitoSOX Red and CellROX Orange Reagent, respectively. Cytoplasmic and secreted metabolites were measured by LC/MS metabolomics.

Results

Treating ARPE-19 with TGFβ2 (10 ng/ml) induced defects in mitochondrial network integrity with increased sphericity and fragmentation. Correspondingly, TGFβ2 reduced both citrate synthase activity and intracellular ATP content and increased expression of mitochondrial dynamics genes (FIS1, MFN1, MFN2). TGFβ2 reduced mitochondrial OXPHOS levels consistent with reduced NDUFB5, a key gene of Complex I of the electron transport chain. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic enzyme genes (PFKFB3, PKM2, LDHA) and glycolytic reserve (Seahorse XF24 Glycolytic Stress Test). Metabolomic profiling revealed changes in the glutamine alpha-ketoglutarate metabolic pathway with TGFβ2 treatment. TGFβ2 induced an accumulation of mitochondrial and cellular ROS that was associated with an upregulation in NOX4 gene expression and concomitant suppression of the mitochondrial antioxidant SOD2.

Conclusions

Our data show that EMT is accompanied by mitochondrial dysfunction, increased oxidative stress and a profound metabolic shift towards reduced OXPHOS and increased glycolysis. Targeting mitochondrial dysfunction and metabolic rewiring is a promising therapeutic strategy for the treatment of fibrotic retinal diseases.