Dataset Information

Crosstalk between Long-Term Sublethal Oxidative Stress and Detrimental Inflammation as Potential Drivers for Age-Related Retinal Degeneration.

ABSTRACT: Age-related retinal degenerations, including age-related macular degeneration (AMD), are caused by the loss of retinal pigmented epithelial (RPE) cells and photoreceptors. The pathogenesis of AMD, deeply linked to the aging process, also involves oxidative stress and inflammatory responses. However, the molecular mechanisms contributing to the shift from healthy aging to AMD are still poorly understood. Since RPE cells in the retina are chronically exposed to a pro-oxidant microenvironment throughout life, we simulated in vivo conditions by growing ARPE-19 cells in the presence of 10 ?M H₂O₂ for several passages. This long-term oxidative insult induced senescence in ARPE-19 cells without affecting cell proliferation. Global proteomic analysis revealed a dysregulated expression in proteins involved in antioxidant response, mitochondrial homeostasis, and extracellular matrix organization. The analyses of mitochondrial functionality showed increased mitochondrial biogenesis and ATP generation and improved response to oxidative stress. The latter, however, was linked to nuclear factor-?B (NF-?B) rather than nuclear factor erythroid 2-related factor 2 (Nrf2) activation. NF-?B hyperactivation also resulted in increased pro-inflammatory cytokines expression and inflammasome activation. Moreover, in response to additional pro-inflammatory insults, senescent ARPE-19 cells underwent an exaggerated inflammatory reaction. Our results indicate senescence as an important link between chronic oxidative insult and detrimental chronic inflammation, with possible future repercussions for therapeutic interventions.

SUBMITTER: Macchioni L

PROVIDER: S-EPMC7823845 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Crosstalk between Long-Term Sublethal Oxidative Stress and Detrimental Inflammation as Potential Drivers for Age-Related Retinal Degeneration.

Macchioni Lara L Chiasserini Davide D Mezzasoma Letizia L Davidescu Magdalena M Orvietani Pier Luigi PL Fettucciari Katia K Salviati Leonardo L Cellini Barbara B Bellezza Ilaria I

Antioxidants (Basel, Switzerland) 20201229 1

Age-related retinal degenerations, including age-related macular degeneration (AMD), are caused by the loss of retinal pigmented epithelial (RPE) cells and photoreceptors. The pathogenesis of AMD, deeply linked to the aging process, also involves oxidative stress and inflammatory responses. However, the molecular mechanisms contributing to the shift from healthy aging to AMD are still poorly understood. Since RPE cells in the retina are chronically exposed to a pro-oxidant microenvironment throu ...[more]

PMID: 33383836

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Project description:The first goal of the study was to find the genes that explain the difference in progression of age-related retinal degeneration (ageRD) between the inbred strains C57BL/6J-c2J (B6a) and BALB/cByJ (C). The specific goal was to find the candidate genes that explain the chromosome 6 and chromosome 10 quantitative trait loci (QTL) found in a previous study. Genes differentially expressed in retina between both strains and that map to the QTL are candidate genes. By integrating different approaches: microarray study, knowledge-based candidate gene search and screening of genetic variants, we found 35 candidate genes for chromosome 6 QTL and 14 candidate genes for chromosome 10 QTL. Five genes for each QTL were selected for sequencing and qPCR analysis. One gene, Tnfaip3 was selected for phenotypic testing and was excluded as the quantitative trait gene for chr10 QTL. The second goal of the study was to find biological processes and pathways associated with ageRD by Gene Ontology analysis of the microarray data. We concentrated on GO terms overrepresented in either strain at 8 months; the age when there is a difference in the phenotype (outer nuclear layer thickness). The GO analysis revealed that transcripts associated with inflammation, mitochondrial envelope, DNA repairing and oxidative stress were increased in the ageRD sensitive C strain with age (8 months). In the ageRD resistant B6a strain transcripts associated with antioxidant response, regulation of blood vessel size and growth factor activity were increased with age. Transcripts associated with cell remodeling and lipid metabolism were increased in either strain with age. This study aims to identify gene expression differences in posterior eyecups between the strains C and B6a at 4 months and at 8 months. Twelve animals were used per strain and time point for a total of 48 mice. Total RNA for 3 animals (6 eyes) were pooled giving a total of 4 biological replicates per strain and time point. The 4-month C samples labeled with Cy5 were hybridized with the 4-month B6a samples labeled with Cy3 (4 biological replicates); the 8-month C samples labeled with Cy5 were hybridized with the 8-month B6a samples labeled with Cy3, for 8 arrays (4 biological replicates). A second set of eight arrays were used to analyzed the same RNA samples but with the dye orientation reversed, yielding a total of 16 arrays in the experiment. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.

Project description:PurposeOxidative injury is involved in retinal and macular degeneration. We aim to assess if retinal degeneration associated with genetic defect modulates the retinal threshold for encountering additional oxidative challenges.MethodsRetinal oxidative injury was induced in degenerating retinas (rd10) and in control mice (WT) by intravitreal injections of paraquat (PQ). Retinal function and structure was evaluated by electroretinogram (ERG) and histology, respectively. Oxidative injury was assessed by immunohistochemistry for 4-Hydroxy-2-nonenal (HNE), and by Thiobarbituric Acid Reactive Substances (TBARS) and protein carbonyl content (PCC) assays. Anti-oxidant mechanism was assessed by quantitative real time PCR (QPCR) for mRNA of antioxidant genes and genes related to iron metabolism, and by catalase activity assay.ResultsThree days following PQ injections (1 µl of 0.25, 0.75, and 2 mM) the average ERG amplitudes decreased more in the WT mice compared with the rd10 mice. For example, following 2 mM PQ injection, ERG amplitudes reduced 1.84-fold more in WT compared with rd10 mice (p = 0.02). Injection of 4 mM PQ resulted in retinal destruction. Altered retina morphology associated with PQ was substantially more severe in WT eyes compared with rd10 eyes. Oxidative injury according to HNE staining and TBARS assay increased 1.3-fold and 2.1-fold more, respectively, in WT compared with rd10 mice. At baseline, prior to PQ injection, mRNA levels of antioxidant genes (Superoxide Dismutase1, Glutathione Peroxidase1, Catalase) and of Transferrin measured by quantitative PCR were 2.1-7.8-fold higher in rd10 compared with WT mice (p<0.01 each), and catalase activity was 1.7-fold higher in rd10 (p = 0.0006).ConclusionsThis data suggests that degenerating rd10 retinas encounter a relatively lower degree of damage in response to oxidative injury compared with normal retinas. Constitutive up-regulation of the oxidative defense mechanism in degenerating retinas may confer such relative protection from oxidative injury.

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Crosstalk between Long-Term Sublethal Oxidative Stress and Detrimental Inflammation as Potential Drivers for Age-Related Retinal Degeneration.

Publications

Crosstalk between Long-Term Sublethal Oxidative Stress and Detrimental Inflammation as Potential Drivers for Age-Related Retinal Degeneration.

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