Cigarette smoke extract and palmitic acid exposed patient-derived iPS- RPE cells as a model for AMD

Abstract

Purpose: Age-related macular degeneration (AMD) is a multifactorial disease in which dysfunction and loss of retinal pigment epithelial (RPE) cells contribute to disease progression. Besides genetics, cigarette smoking and high fat diet are important environmental and metabolic risk factors for AMD and are associated with RPE dysfunction. However, the effects of combined environmental and metabolic stress on AMD-derived RPE cells remain poorly understood. In this study, we used patient-derived induced pluripotent stem cell-derived RPE (iPSC-RPE) cells to examine the effects of cigarette smoke extract (CSE) and palmitic acid (PA) on RPE cell function. Methods: iPSCs derived from non-AMD and AMD donors were differentiated into RPE cells and characterized based on morphology, RPE-specific marker expression, polarity, gene expression, and barrier function. Mature iPSC-RPE cells were exposed to different concentrations of CSE and PA to determine experimental conditions. Cell death, epithelial-to-mesenchymal transition (EMT)-like morphological changes, stress-related gene expression, RPE polarity, TEER, VEGF-A and PEDF secretion, and intracellular lipid accumulation were then evaluated following CSE, PA, or combined CSE and PA exposure. Results: Differentiated iPSC-RPE cells developed typical pigmented, hexagonal morphology, expressed RPE-specific markers, and formed polarized monolayers. Both CSE and PA increased cell death in a dose-dependent manner in the control and AMD-iPSC-RPE, with AMD-iPSC-RPE exhibiting greater susceptibility, particularly those from patients with dry AMD. CSE and PA also induced also induced EMT-like morphological changes and increased the expression of ZEB1, NQO1, and IL8. Furthermore, CSE and PA disrupted the polarized distribution of ZO-1 and Best1, significantly reduced the polarity ratio, decreased PEDF and VEGF-A secretion and increased intracellular lipid accumulation. Combined CSE and PA treatment produced more pronounced effects than either treatment alone in AMD lines. Conclusions: AMD- iPSC-RPE cells, particularly those derived from dry AMD donors, exhibit increased susceptibility to environmental and metabolic stress. Exposure to CSE and PA revealed this vulnerability through alterations in cell survival, stress responses, epithelial morphology, polarity, secretory function, and intracellular lipid accumulation. These findings highlight the effects of CSE and lipid stress on RPE metabolism and underscore the utility of patient-derived iPSC-RPE cells as a model for investigating how environmental and metabolic factors contribute to RPE dysfunction and AMD pathogenesis.