Epithelial–mesenchymal transition (EMT) and endothelial–mesenchymal transition (EndMT) are physiological processes required for normal embryogenesis. However, these processes can be hijacked in pathological conditions to facilitate tissue fibrosis and cancer metastasis. In the eye, EMT and EndMT play key roles in the pathogenesis of subretinal fibrosis, the end-stage of age-related macular degeneration (AMD) that leads to profound and permanent vision loss. Predominant in subretinal fibrotic lesions are matrix-producing mesenchymal cells believed to originate from the retinal pigment epithelium (RPE) and/or choroidal endothelial cells (CECs) through EMT and EndMT, respectively. Recent evidence suggests that EMT of RPE may also be implicated during the early stages of AMD. Transforming growth factor-beta (TGFβ) is a key cytokine orchestrating both EMT and EndMT. Investigations in the molecular mechanisms underpinning EMT and EndMT in AMD have implicated a myriad of contributing factors including signaling pathways, extracellular matrix remodelling, oxidative stress, inflammation, autophagy, metabolism and mitochondrial dysfunction. Questions arise as to differences in the mesenchymal cells derived from these two processes and their distinct mechanistic contributions to the pathogenesis of AMD. Detailed discussion on the AMD microenvironment highlights the synergistic interactions between RPE and CECs that may augment the EMT and EndMT processes in vivo. Understanding the differential regulatory networks of EMT and EndMT and their contributions to both the dry and wet forms of AMD can aid the development of therapeutic strategies targeting both RPE and CECs to potentially reverse the aberrant cellular transdifferentiation processes, regenerate the retina and thus restore vision.

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFβ2) suppressed PGC-1α expression. Correspondingly, TGFβ2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.

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, PFKL, 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. Support or Funding Information: 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

Mitochondrial dysfunction and oxidative stress play a central role in the onset and progression of numerous age‐associated retinal pathologies such as AMD. Our previous work indicates that the master metabolic regulators, PGC‐1α and PGC‐1β, exert contrasting functions in RPE homeostasis and implicates PGC‐1β as a novel disease‐associated target in AMD. In this study, we characterized the molecular regulation and biological consequences of pathological PGC‐1β induction in RPE. First, we show that PGC‐1β is specifically induced in RPE by AMD‐associated pro‐oxidative and pro‐inflammatory stimuli. NF‐kB is a primary regulator of RPE stress response known to promote RPE dysfunction in AMD. Activation of the NF‐κB pathway by TNFα promotes PGC‐1β expression in RPE. NF‐κB‐dependent transcriptional induction of PGC‐1β was abrogated by silencing of the RelB/NF‐κB non canonical pathway. In order to evaluate the effect of PGC‐1β induction at physiological levels observed in vitro and in humans, we developed an adenoviral Tet‐on expression system able to tightly control PGC‐1β overexpression in RPE. Comparative analysis of PGC‐1β expressing cells (PGC1B‐Dox+) and controls (PGC1B‐Dox− and GFP‐Dox+) shows that physiological PGC‐1β induction promotes mitoROS production and reduces the cells’ anti‐oxidative capacity under both basal and challenged conditions. Conversely, we demonstrate that PGC‐1β silencing protects cells from mitochondrial targeting pro‐oxidants. As recent transcriptome analysis show that PGC‐1β (but not PGC‐1α) expression is increased in neovascular AMD patients compared to unaffected controls, we examined PGC‐1β pro‐angiogenic effect in choroidal neovascularization. Using an ex vivo choroid explant sprouting model, we demonstrate that gene augmentation of PGC1β in RPE significantly enhances choroidal vessels sprouting. A similar pro‐angiogenic effect was observed when explants were exposed to conditioned media from PGC1B‐Dox+ cells but not from PGC1B‐Dox− or GFP‐Dox+ cells. Protein array analysis indicates that PGC‐1β increases the secretion by RPE of pro‐angiogenic factors (PlGF, FGF2, Leptin, HB‐EGF) while repressing anti‐angiogenic ones (Thrombospondin, Endostatin, Angiostatin). Finally, we confirmed our in vitro data by overexpressing PGC‐1β in the RPE of adult mice using a lentiviral delivery method. RPE‐specific expression of PGC‐1β promotes a rapid disorganization and dysfunction of the RPE leading to spontaneous formation of neovascular lesions in the subretinal space. These findings highlight PGC‐1β as a major regulator of RPE stress response and validate human data associating PGC‐1β induction in RPE to neovascular AMD. Support or Funding Information: This work was funded by donations to the Macular Degeneration Research, a program of the BrightFocus Foundation (MSG), the Grimshaw‐Gudewicz Charitable Foundation (MSG), the Iraty Award (MSG), the China Scholarship Council (SC), and the NEI Core Grant P30EYE003790.