Dataset Information

Complement 3a Mediates CCN2/CTGF in Human Retinal Pigment Epithelial Cells.

ABSTRACT: Complement 3 (C3) is the crucial component of the complement cascade when retina was exposed to external stimulus. Cellular communication network 2/connective tissue growth factor (CCN2/CTGF) is important in response of retinal stress and a fulcrum for angiogenesis and fibrosis scar formation. Our study aims to explore the interaction between C3 and CCN2/CTGF via bioinformatics analyses and in vitro cell experiments. The GSE dataset was selected to analyse the chemokine expression in human retinal pigment epithelium (ARPE-19) cells under stimulus. Then, RPE cells were further transfected with or without C3 siRNA, followed by C3a (0.1 μM or 0.3 μM) for 24, 48, and 72 hours. Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to measure CCN2/CTGF mRNA and protein levels. The GSE36331 revealed C3 expression was significantly elevated in RPE under stimulus. Compared with negative control, CCN2/CTGF mRNA was increased with all types of C3a treatments, whereas a significant increase of protein level was only observed with high concentration of 0.3 μM C3a for a prolonged 72-hour time. Compared with nontransfected cells, significant reductions of CCN2/CTGF mRNA were observed in the C3 siRNA transfected cells with 0.3 μM C3a for 24, 48, and 72 hours, and a significant reduction of CCN2/CTGF protein was observed with 0.3 μM C3a for 48 hours. C3 was elevated in RPE under environmental stimulus and long-term exposure to specified concentration of C3a increased CCN2/CTGF expression in RPE, which could be partially reversed by C3 siRNA.

SUBMITTER: Xiao K

PROVIDER: S-EPMC9581697 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Complement 3a Mediates CCN2/CTGF in Human Retinal Pigment Epithelial Cells.

Xiao Kang K Xu Zhiyan Z Chen Zhengyu Z Long Qin Q Chen Youxin Y

Journal of ophthalmology 20220730

<h4>Background</h4>Complement 3 (C3) is the crucial component of the complement cascade when retina was exposed to external stimulus. Cellular communication network 2/connective tissue growth factor (CCN2/CTGF) is important in response of retinal stress and a fulcrum for angiogenesis and fibrosis scar formation. Our study aims to explore the interaction between C3 and CCN2/CTGF via bioinformatics analyses and <i>in vitro</i> cell experiments.<h4>Methods</h4>The GSE dataset was selected to analys ...[more]

PMID: 36276919

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Project description:PurposePhotoreceptor degeneration occurs in various retinal diseases including age-related macular degeneration (AMD), Retinitis pigmentosa (RP), and diabetic retinopathy (DR). However, molecular mechanisms are not fully understood yet. The retinal pigment epithelium (RPE) forms the outer blood retinal barrier (oBRB) and supplies glucose, oxygen and nutrients from the fenestrated choriocapillaris to photoreceptors for visual function. Therefore, RPE dysfunction leads to photoreceptor injury/death and progression of blinding eye diseases. This study aims to understand the role of the thioredoxin (Trx) and its reductase (TrxR) redox signaling in human RPE dysfunction and cell death mechanism(s) in an in vitro system.MethodsA human RPE cell line (APRE-19) was cultured in DMEM/F12 medium and treated with auranofin (AF - 4 μM, an inhibitor of TrxR) for 4 and 24 h. Mitochondrial and lysosomal function, cellular oxidative stress and NLRP3 inflammasome activity were measured using cell assays, Western blotting, and confocal microscopy. Antioxidants and anti-inflammatory compounds were tested for blocking AF effects on RPE damage. Cell death mechanisms (LDH release to culture media) were determined using necroptosis, ferroptosis and pyroptosis inhibitors. P < 0.05 was considered significant in statistical analysis.ResultsAuranofin causes mitochondrial dysfunction (Δψm↓ and ATP↓), oxidative stress (H2O2↑) and mitophagic flux to lysosomes. Furthermore, the lysosomal enzyme (cathepsin L) activity is reduced while that of pro-inflammatory caspase-1 (NLRP3 inflammasome) is enhanced in ARPE-19. These effects of AF on ARPE-19 are inhibited by antioxidant N-acetylcysteine (5 mM, NAC) and significantly by a combination of SS31 (mitochondrial antioxidant) and anti-inflammatory drugs (amlexanox and tranilast). AF also causes cell death as measured by cytosolic LDH release/leakage, which is not inhibited by either ferrostatin-1 or necrostatin-1 (ferroptosis and necroptosis inhibitors, respectively). Conversely, AF-induced LDH release is significantly reduced by MCC950 and Ac-YVAD-cmk (NLRP3 and Caspase-1 inhibitors, respectively), suggesting a pro-inflammatory cell death by pyroptosis.ConclusionThe Trx/TrxR redox system is critical for RPE function and viability. We previously showed that thioredoxin-interacting protein (TXNIP) is strongly induced in DR inhibiting the Trx/TrxR system and RPE dysfunction. Therefore, our results suggest that the TXNIP-Trx-TrxR redox pathway may participate in RPE dysfunction in DR and other retinal neurodegenerative diseases.

Project description:BackgroundCurcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro.Methodology/principal findingsCellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein.ConclusionIt is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (?10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of cancer or in the treatment of eye diseases, retinal function should be monitored carefully.

Project description:RationaleAge-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in the developed world. Aging, inflammation and complement dysregulation affecting the retinal pigment epithelium (RPE), are considered significant contributors in its pathogenesis and several evidences have linked tumor necrosis factor alpha (TNF-α) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown to have cytoprotective effects in human clinical trials as well as having anti-inflammatory and anti-vascular exudative effects in animals. The purpose of this study was to evaluate if acadesine is able to suppress TNF-α induced C3 in RPE cells.MethodsARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-α was used for C3 induction in the presence or absence of acadesine. Small molecule inhibitors and siRNA were used to determine if acadesine exerts its effect via the extracellular or intracellular pathway and to evaluate the importance of AMPK for these effects. The expression level of C3 was determined by immunoblot analysis.ResultsAcadesine suppresses TNF-α induced C3 in a dose dependent manner. When we utilized the adenosine receptor inhibitor dipyridamole (DPY) along with acadesine, acadesine's effects were abolished, indicating the necessity of acadesine to enter the cell in order to exert it's action. However, pretreatment with 5-iodotubericidin (5-Iodo), an adenosine kinase (AK) inhibitor, didn't prevent acadesine from decreasing TNF-α induced C3 expression suggesting that acadesine does not exert its effect through AMP conversion and subsequent activation of AMPK. Consistent with this, knockdown of AMPK α catalytic subunit did not affect the inhibitory effect of acadesine on TNF-α upregulation of C3.ConclusionsOur results suggest that acadesine suppresses TNF-α induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.

Dataset Information

Complement 3a Mediates CCN2/CTGF in Human Retinal Pigment Epithelial Cells.

Publications

Complement 3a Mediates CCN2/CTGF in Human Retinal Pigment Epithelial Cells.

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