Modeling the Dynamic AMD-Associated Chronic Oxidative Stress Changes in Human ESC and iPSC-Derived RPE Cells

Abstract

Citation: Garcia TY, Gutierrez M, Reynolds J, Lamba DA. Modeling the dynamic AMD-associated chronic oxidative stress changes in human ESC and iPSC-derived RPE cells. Invest Ophthalmol Vis Sci. 2015;56:7480-7488. DOI:10.1167/iovs.15-17251 PURPOSE. Here we use human embryonic stem cells (hESCs) and human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells to model chronic oxidative stress in vitro. This model allows us to understand the evolution of chronic stress response in RPE in vivo, as well as to monitor microRNAs changes. Finally, we use this in vitro model to identify a partial agonist of NRF2 that is protective against reactive oxygen species (ROS)-induced cytotoxicity.METHODS. The hESCs and hiPSCs were differentiated toward an RPE fate. Upon maturation, RPE cells were subjected to chronic oxidative stress using Paraquat (PQ). The cells were then analyzed using immunocytochemistry and quantitative RT-PCR to look for changes in gene expression and microRNA changes. Small molecules targeting NRF2 pathways were utilized to look for protection against oxidative stress-induced apoptosis.RESULTS. We show that 160 lM PQ can be used to generate a model of chronic oxidative stress in RPE cells derived from hESCs and hiPSCs. Using this model, we characterize the NRF2 pathway effectors during the early and late stages of chronic oxidative stress and identify microRNAs changes during oxidative stress. We find that hsa-miR144 modulates NRF2 activity during ROS stress. Lastly, we found a small molecule modulator of NRF2 that plays a protective role against oxidative stress-induced RPE apoptosis.CONCLUSIONS. In summary, pluripotent stem cell-derived retinal cells can be used to model retinal diseases in a dish. This can provide an unprecedented opportunity to understand the evolution of disease processes and allow us to identify novel therapeutics.Keywords: age-related macular degeneration, disease modeling, oxidative stress, microRNA, NRF2 A ge-related macular degeneration (AMD) is the leading cause of worldwide blindness in the elderly, affecting almost 15 million people in the United States. Retinal changes associated with AMD are present in approximately 10% of people over 65 years of age and as many as one in three people over the age of 80 years. Although the disease was first described in the 1800s, its etiology remains poorly understood, and multiple factors may be involved in the progression of the disorder, including chronic oxidative stress. A number of studies have associated oxidative stress as the key driver to AMD.1,2 The retina is one of the tissues with the greatest consumption of oxygen in the body. 3 This results in significant production of reactive oxygen species (ROS) in the retinal pigment epithelium (RPE). Increasing age results in the loss of ability to deal with this excessive ROS, which leads to oxidative damage.There are no known effective forms of treatment for the common dry form of AMD. This likely is due to the complex multifactorial etiology of AMD and...

An Eye on Age-Related Macular Degeneration: The Role of MicroRNAs in Disease Pathology

“…Importantly, the miRNA with the highest number of studies supporting its involvement in AMD is miR-146a. Two studies performed in patients, and three studies performed in human RPE cells or in mouse retinae revealed a miR-146a upregulation, although one study indicated this miRNA species to be downregulated after oxygen-induced retinopathy (OIR) [56, 57, 71, 73–75]. Although technically not an AMD model, OIR is a well-established protocol used to study hyperoxia-induced retinal neovascularization and inflammation in mice, two well-known characteristics of AMD [76, 77].…”

“…b Plasma[103]miR-374ahsa-miR-374a-5p0.1ARPE-19 cellsOxidative stress[82]hsa-miR-374a-5p−12.92Plasma[103]miR-424mmu-miR-424 a +Mouse retinaOIR[90]hsa-miR-424-5p−0.338Plasma[51]hsa-miR-424-5p3.97; 3.28Serum[52]

FC fold change , AMD age-related macular degeneration , miRNA micro RNA , GA AMD graphic atrophy AMD , RPE retinal pigment epithelium, OIR oxygen-induced retinopathy, VH vitreous humor, pro - inf pro-inflammatory, ARPE - 19 adult retinal pigment epithelium cell line

a Mature miRNA strand unknown/not mentioned

b o.p. : miRNA was only expressed in the patient group c Oxidative stress was induced using H 2 O 2 [82] or Paraquat [73] d A “+” indicates miRNA upregulated/higher levels and “−” indicates miRNA downregulated/lower levels

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“…: miRNA was only expressed in the patient group c Oxidative stress was induced using H 2 O 2 [82] or Paraquat [73] d A “+” indicates miRNA upregulated/higher levels and “−” indicates miRNA downregulated/lower levels…”

“…All words with an occurrence greater than 4 are plotted and their relative abundance is indicated by the size of the respective word. Figure was plotted with Tagxedo (http://www.tagxedo.com/app.html) using words from the abstracts of 21 publications related to miRNAs in AMD [51, 52, 56, 57, 71, 73–75, 77, 82, 89, 90, 99, 103–110] …”

Proteome and Secretome Dynamics of Stem Cell-Derived Retinal Pigmented Epithelium in Response to Acute and Chronic ROS

“…Chronic oxidative damage in RPE may increase the expression of proteins (see 10 ) and probably causes apoptosis. Stem cellderived RPE was previously used as a model system with chronic, low-level oxidative stress from paraquat (PQ) to study the NRF2-mediated transcriptional responses 11 . PQ is well tolerated by RPE 12 and is a suitable mimic for pathological oxidative stress that occurs in AMD, which is thought to be mostly generated in mitochondria 13 .…”