Dataset Information

Regulation of ABCA1 by AMD-Associated Genetic Variants and Hypoxia in iPSC-RPE.

ABSTRACT: Age-related macular degeneration (AMD) is a progressive disease of the macula characterized by atrophy of the retinal pigment epithelium (RPE) and photoreceptor degeneration, leading to severe vision loss at advanced stages in the elderly population. Impaired reverse cholesterol transport (RCT) as well as intracellular lipid accumulation in the RPE are implicated in AMD pathogenesis. Here, we focus on ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transport protein in the RPE, and analyze conditions that lead to ABCA1 dysregulation in induced pluripotent stem cell (iPSC)-derived RPE cells (iRPEs). Our results indicate that the risk-conferring alleles rs1883025 (C) and rs2740488 (A) in ABCA1 are associated with increased ABCA1 mRNA and protein levels and reduced efficiency of cholesterol efflux from the RPE. Hypoxia, an environmental risk factor for AMD, reduced expression of ABCA1 and increased intracellular lipid accumulation. Treatment with a liver X receptor (LXR) agonist led to an increase in ABCA1 expression and reduced lipid accumulation. Our data strengthen the homeostatic role of cholesterol efflux in the RPE and suggest that increasing cellular cholesterol export by stimulating ABCA1 expression might lessen lipid load, improving RPE survival and reducing the risk of developing AMD.

SUBMITTER: Peters F

PROVIDER: S-EPMC8953808 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Regulation of ABCA1 by AMD-Associated Genetic Variants and Hypoxia in iPSC-RPE.

Peters Florian F Ebner Lynn J A LJA Atac David D Maggi Jordi J Berger Wolfgang W den Hollander Anneke I AI Grimm Christian C

International journal of molecular sciences 20220316 6

Age-related macular degeneration (AMD) is a progressive disease of the macula characterized by atrophy of the retinal pigment epithelium (RPE) and photoreceptor degeneration, leading to severe vision loss at advanced stages in the elderly population. Impaired reverse cholesterol transport (RCT) as well as intracellular lipid accumulation in the RPE are implicated in AMD pathogenesis. Here, we focus on ATP-binding cassette transporter A1 (ABCA1), a major cholesterol transport protein in the RPE, ...[more]

PMID: 35328615

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Project description:IntroductionAge-related macular degeneration (AMD), a degenerative disease of the macula, is caused by an interplay of diverse risk factors (genetic predisposition, age and lifestyle habits). One of the main genetic risks includes the Y402H polymorphism in complement Factor H (FH), an inhibitor of complement system activation. There has been, and continues to be, much discussion around the functional consequences of this Y402H polymorphism, whether the soluble FH protein confers its risk association, or if the cells expressing the protein themselves are affected by the genetic alteration. In our study, we examined the cell characteristics of the retinal pigment epithelium (RPE) cells, which play a major role in retinal homeostasis and stability and which are synonymously linked to AMD.MethodsHere, we employ RPE cells derived from induced pluripotent stem cells (iPSC) generated from donors, carrying either homozygous 402Y (low risk) or 402H (high risk) variants of the CFH gene. RPE cells were treated with Hydroquinone (HQ), a component of cigarette smoke, to induce oxidative damage.ResultsIntriguingly, RPE cells carrying high genetic risk proved more vulnerable to oxidative insult when exposed to HQ, as demonstrated by increased cytotoxicity and caspase activation, compared to the low-risk RPE cells. The exposure of RPE cells to RPE conditioned medium, normal human serum (NHS) and inactivated NHS (iNHS) had minimal impact on cell cytotoxicity and caspase activation, nor did the presence of purified soluble FH rescue the observed effects. Considering the known connection of oxidative stress to proteotoxic stress and degrading processes, we investigated the unfolded protein response (UPR) and autophagy. When exposed to HQ, RPE cells showed an increase in autophagy markers; however, iPSC-RPE cells carrying high genetic risk showed an overall reduced autophagic flux.DiscussionOur findings suggest that the degree of cellular susceptibility to oxidative stress is not conferred by soluble FH protein and other complement sources, but intercellularly because of the corresponding genetic risk predisposition. Our data support the hypothesis that RPE cells carrying high genetic risk are less resilient to oxidative stress.

Project description:BackgroundAge-related macular degeneration (AMD) is a multifactorial chronic disease of the eye. Several candidate pathways have been hypothesized to play a role in AMD pathogenesis. Our work and those of others suggests inflammasome activity as a mechanism associated with retinal pigment epithelial (RPE) cell demise. X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptosis factor, has recently been shown to regulate inflammasome activity in non-ocular cells. The purpose of this study is to characterize XIAP's regulatory role in RPE.MethodsProtein lysates of eye tissues from rats (vinpocetine- or aurin tricarboxylic acid complex-treated, ATAC, vs naïve) and mice (wild type vs Caspase-4-/-) were utilized to analyze XIAP protein levels. Immunohistochemistry was used to detect NLRP3 levels in the RPE layer. In vitro inflammasome activation on RPE cells was achieved with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) stimulation. Levels of XIAP mRNA and 18S RNA were quantified by RT-PCR. Cell culture supernatants were tested directly for secreted IL-1β by ELISA or concentrated for the detection of secreted IL-18 by western blot. Protein lysates from RPE in cell culture were collected for the measurement of cleaved caspase-1 p20, XIAP, and GAPDH. Data are presented as Mean ± SD. p < 0.05 is considered statistically significant.ResultsThe XIAP protein level was significantly increased when the inflammasome was inhibited at the "activation" step by ATAC, but not the "priming" step, in vivo. Concomitantly, NLRP3 immunoreactivity was lower in the RPE layer of animals fed with ATAC. In mice where caspase-1 cleavage was impaired by the genetic deficiency in caspase-4, the XIAP protein level increased in eye tissues. In RPE cell culture, Leu-Leu-OMe stimulation led to caspase-1 cleavage, cytokine secretion, and XIAP reduction, which can be abolished by Z-YVAD-FMK. When XIAP siRNA was given as a pre-treatment to RPE in vitro, Leu-Leu-OMe induced IL-1β/IL-18 secretion was enhanced, whereas overexpressing XIAP reduced IL-1β secretion under inflammasome activation, both compared to controls cells.ConclusionsTogether, these data suggest XIAP-mediated inhibition of inflammasome activity in RPE may provide insights into the biological consequences of inflammasome activation in RPE and reveals the caspase-1/XIAP/IL-1β/IL-18 axis as a target for broader applications in AMD biology and treatment design.

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Regulation of ABCA1 by AMD-Associated Genetic Variants and Hypoxia in iPSC-RPE.

Publications

Regulation of ABCA1 by AMD-Associated Genetic Variants and Hypoxia in iPSC-RPE.

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