Pentosan polysulfate (PPS) is a semisynthetic sulfated polysaccharide that was approved by the United States Food and Drug Administration (FDA) for treatment of interstitial cystitis (IC). A 2018 study by our group described a vision-threatening macular toxicity associated with long-term use of PPS. However, given the relatively recent characterization of PPS maculopathy, we have limited knowledge of its pathophysiology. The present study therefore investigated the pathophysiology of PPS maculopathy in a cell culture model, assessing impacts of PPS exposure on morphology and mitochondrial function. We treated ARPE-19 cells with increasing doses of PPS and investigated both mitoprotective and cytoprotective mechanisms, mitochondrial reactive oxygen species production (ROS) and respiration, cellular structure, and retinal pigment epithelium (RPE) dysfunction through phagocytosis assays. We found that PPS increased mitochondrial superoxide accumulation and that increased doses of PPS impaired basal and maximal respiration in a Seahorse assay without the expected response of increases in the cellular energy sensor pAMPK. PPS exposure disrupted mitochondrial and cell protective mechanisms against ROS accumulation as assessed through examination of mitochondrial biogenesis markers PGC-1α and SIRT1 and autophagy markers LC3 and p62. PINK1 expression increased with increasing duration of exposure to PPS. Further, we found that PPS led to functional and structural changes to RPE cells, which exhibited an increase in cell aspect ratio and impaired phagocytosis with higher doses of PPS. Lastly, we found an increase in cell death in response to higher doses of PPS, evident through ethidium homodimer cell viability assays. Taken together, our study shows PPS exposure has profound effects on RPE viability and function through impairment of mitochondrial respiration and mito- and cyto-protective mechanisms and highlights mitochondrial insult as a potential focus of future PPS research.

Age-related macular degeneration (AMD) is a progressive complex eye disease and one of the leading causes of blindness. AMD progression is marked by molecular changes in the retinal pigmented epithelium (RPE) which include increased reactive oxygen species (ROS) accumulation, mitochondrial dysfunction - eventually leading to dysfunctional RPE. Mitophagy regulator, Pink1, is reduced in the RPE of AMD patients and Pink1 loss leads to a shift from mitochondrial respiration to glycolysis. Serine is a non-essential amino acid which is de novo synthesized from glycolytic intermediate 3-PG via the rate limiting enzyme PHGDH. Serine is tightly integrated into anabolic processes like glutathione (GSH) cycling, maintaining NADH/NADPH pools leading to changes in AMPK signaling. Here, we show that Pink1 loss leads to a reduction in PHGDH and serine levels in the RPE leading to impaired mitochondrial structure and function, increased ROS mediated damage, increased inflammation, and hampered retinal function. Serine supplementation rescued ROS accumulation, balanced GSH abundance, and increased retinal function. Overall, our study highlights the potential of dietary serine in ROS management in AMD.

Dry age-related macular degeneration (AMD) is a leading cause of blindness in the elderly, and treatment options are limited. Cigarette smoke (CS), the primary modifiable environmental risk factor for AMD, damages the retinal pigment epithelium (RPE) by inducing oxidative stress, which in turn activates multiple stress response pathways. Low levels of stress induce autophagy, a cell-recycling mechanism; moderate stress induces epithelial-mesenchymal transition (EMT); and high, sustained stress, once other cytoprotective pathways are exhausted, leads to cell death. All three pathways are implicated in AMD pathogenesis. Although CS is known to induce oxidative stress, its impact on these distinct stress response pathways remains understudied, and few studies have directly compared mouse and human RPE responses to the same stressor. Here, we examined the effect of varying concentrations and durations of cigarette smoke condensate (CSC) exposure on oxidative stress, autophagy, EMT, and cell death in ARPE-19 (human) and mouse RPE (MRPE) cells. Understanding which stress response pathways predominate at which CSC exposure levels may help guide the development of AMD treatments stratified by smoking history.

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.