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Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y₂ receptor signaling.

ABSTRACT: Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O₂ atmosphere and addition of CoCl₂, respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1? levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O₂ induced expression of NLRP3 and pro-IL-1? genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1? proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y₂ and adenosine A₁ receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y₂ (but not A₁) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y₂ receptor signaling.

SUBMITTER: Doktor F

PROVIDER: S-EPMC6298930 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y<sub>2</sub> receptor signaling.

Doktor Fabian F Prager Philipp P Wiedemann Peter P Kohen Leon L Bringmann Andreas A Hollborn Margrit M

Purinergic signalling 20181110 4

Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) ...[more]

PMID: 30415294

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Project description:PurposeVariants of complement factor genes, hypoxia and oxidative stress of the outer retina, and systemic hypertension affect the risk of age-related macular degeneration. Hypertension often results from the high intake of dietary salt that increases extracellular osmolarity. We determined the effects of extracellular hyperosmolarity, hypoxia, and oxidative stress on the expression of complement genes in cultured (dedifferentiated) human RPE cells and investigated the effects of C9 siRNA and C9 protein on RPE cells.MethodsHyperosmolarity was induced by adding 100 mM NaCl or sucrose to the culture medium. Hypoxia was induced by culturing cells in 1% O2 or by adding the hypoxia mimetic CoCl2. Oxidative stress was induced by adding H2O2. Gene and protein expression levels were determined with real-time RT-PCR, western blot, and ELISA analyses. The expression of the nuclear factor of activated T cell 5 (NFAT5) and complement factor (C9) was knocked down with siRNA.ResultsExtracellular hyperosmolarity, hypoxia, and oxidative stress strongly increased the transcription of the C9 gene, while the expression of the C3, C5, CFH, and CFB genes was moderately altered or not altered at all. Hyperosmolarity also induced a moderate increase in the cytosolic C9 protein level. The hyperosmotic C9 gene expression was reduced by inhibitors of the p38 MAPK, ERK1/2, JNK, and PI3K signal transduction pathways and of the transcription factors STAT3 and NFAT5. The hypoxic C9 gene expression was reduced by a STAT3 inhibitor. The knockdown of C9 with siRNA decreased the hypoxic vascular endothelial growth factor (VEGF) and NLRP3 gene expression, the hypoxic secretion of VEGF, and the hyperosmotic expression of the NLRP3 gene. Exogenous C9 protein inhibited the hyperosmotic expression of the C9 gene, the hypoxic and hyperosmotic VEGF gene expression, and the hyperosmotic expression of the NLRP3 gene. Both C9 siRNA and C9 protein inhibited inflammasome activation under hyperosmotic conditions, as indicated by the decrease in the cytosolic level of mature IL-1β.ConclusionsThe expression of the C9 gene in cultured RPE cells is highly induced by extracellular hyperosmolarity, hypoxia, and oxidative stress. The data may support the assumption that C9 gene expression may stimulate the expression of inflammatory (NLRP3) and angiogenic growth factors (VEGF) in RPE cells. Extracellular C9 protein may attenuate this effect, in part via negative regulation of the C9 mRNA level.

Project description:With ageing extracellular material is deposited in Bruch's membrane, as drusen. Lipofuscin is deposited in retinal pigment epithelial cells. Both of these changes are associated with age related macular degeneration, a disease now believed to involve chronic inflammation at the retinal-choroidal interface. We hypothesise that these molecules may act as danger signals, causing the production of inflammatory chemokines and cytokines by the retinal pigment epithelium, via activation of pattern recognition receptors.ARPE-19 cells were stimulated in vitro with the following reported components of drusen: amyloid-ß (1-42), Carboxyethylpyrrole (CEP) modified proteins (CEP-HSA), N?-(Carboxymethyl)lysine (CML) modified proteins and aggregated vitronectin. The cells were also stimulated with the major fluorophore of lipofuscin: N-retinylidene-N-retinylethanolamine (A2E). Inflammatory chemokine and cytokine production was assessed using Multiplex assays and ELISA. The mechanistic evaluation of the NLRP3 inflammasome pathway was assessed in a stepwise fashion.Of all the molecules tested only A2E induced inflammatory chemokine and cytokine production. 25 µM A2E induced the production of significantly increased levels of the chemokines IL-8, MCP-1, MCG and MIP-1?, the cytokines IL-1ß, IL-2, IL-6, and TNF-?, and the protein VEGF-A. The release of IL-1ß was studied further, and was determined to be due to NLRP3 inflammasome activation. The pathway of activation involved endocytosis of A2E, and the three inflammasome components NLRP3, ASC and activated caspase-1. Immunohistochemical staining of ABCA4 knockout mice, which show progressive accumulation of A2E levels with age, showed increased amounts of IL-1ß proximal to the retinal pigment epithelium.A2E has the ability to stimulate inflammatory chemokine and cytokine production by RPE cells. The pattern recognition receptor NLRP3 is involved in this process. This provides further evidence for the link between A2E, inflammation, and the pathogenesis of AMD. It also supports the recent discovery of NLRP3 inflammasome activation in AMD.

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Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y₂ receptor signaling.

Publications

Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y<sub>2</sub> receptor signaling.

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Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y2 receptor signaling.

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Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y<sub>2</sub> receptor signaling.

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OmicsDI is part of the ELIXIR infrastructure

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Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y₂ receptor signaling.