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Effects of FTMT Expression by Retinal Pigment Epithelial Cells on Features of Angiogenesis.

ABSTRACT: Aberrant angiogenesis is a pathological feature of a number of diseases and arises from the uncoordinated expression of angiogenic factors as response to different cellular stresses. Age-related macular degeneration (AMD), a leading cause of vision loss, can result from pathological angiogenesis. As a mutation in the mitochondrial ferritin (FTMT) gene has been associated with AMD, its possible role in modulating angiogenic factors and angiogenesis was investigated. FTMT is an iron-sequestering protein primarily expressed in metabolically active cells and tissues with high oxygen demand, including retina. In this study, we utilized the human retinal pigment epithelial cell line ARPE-19, both as undifferentiated and differentiated cells. The effects of proinflammatory cytokines, FTMT knockdown, and transient and stable overexpression of FTMT were investigated on expression of pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial-derived factor (PEDF). Proinflammatory cytokines induced FTMT and VEGF expression, while NF-?B inhibition significantly reduced FTMT expression. VEGF protein and mRNA expression were significantly increased in FTMT-silenced ARPE-19 cells. Using an in vitro angiogenesis assay with endothelial cells, we showed that conditioned media from FTMT-overexpressing cells had significant antiangiogenic effects. Collectively, our findings indicate that increased levels of FTMT inhibit angiogenesis, possibly by reducing levels of VEGF and increasing PEDF expression. The cellular models developed can be used to investigate if increased FTMT may be protective in angiogenic diseases, such as AMD.

SUBMITTER: Buyandelger U

PROVIDER: S-EPMC7279371 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Effects of FTMT Expression by Retinal Pigment Epithelial Cells on Features of Angiogenesis.

Buyandelger Undral U Walker Douglas G DG Yanagisawa Daijiro D Morimura Toshifumi T Tooyama Ikuo I

International journal of molecular sciences 20200521 10

Aberrant angiogenesis is a pathological feature of a number of diseases and arises from the uncoordinated expression of angiogenic factors as response to different cellular stresses. Age-related macular degeneration (AMD), a leading cause of vision loss, can result from pathological angiogenesis. As a mutation in the mitochondrial ferritin (FTMT) gene has been associated with AMD, its possible role in modulating angiogenic factors and angiogenesis was investigated. FTMT is an iron-sequestering p ...[more]

PMID: 32455741

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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:Exosomes are informative microvesicles associated with intercellular communication via the transfer of many molecular constituents such as proteins, lipids, and nucleic acids; environmental changes and the cellular status around cells greatly affect exosome components. Cells of the retinal pigment epithelium (RPE) are key players in retinal homeostasis. Transforming growth factor (TGF)-? and tumour necrosis factor (TNF)-? are increased in the vitreous and retina in several retinal diseases and activate and undergo epithelial-mesenchymal transition (EMT) in RPE cells. EMT is closely associated with mechanisms of wound healing, including fibrosis and related angiogenesis; however, whether exosome components depend on the cell status, epithelium or mesenchyme and whether these exosomes have pro- or anti-angiogenic roles in the retina are unknown. We performed this study to investigate whether these EMT inducers affect the kinds of components in exosomes secreted from RPE cells and to assess their angiogenic effects. Exosomes were collected from culture media supernatants of a human RPE cell line (ARPE-19) stimulated with or without 10 ng/ml TNF-? and/or 5 ng/ml TGF-?2. NanoSight tracking analysis and immunoblot analysis using exosome markers were used to qualify harvested vesicles. Angiogenic factor microarray analysis revealed that exosomes derived from ARPE-19 cells cultured with TNF-? alone (Exo-TNF) and co-stimulated with TNF-? and TGF-?2 (Exo-CO) contained more angiogenic factors than exosomes derived from control cells (Exo-CTL) or ARPE-19 cells cultured with TGF-?2 alone (Exo-TGF). To assess the effect on angiogenesis, we performed chemotaxis, tube formation, and proliferation assays of human umbilical vein endothelial cells (HUVECs) stimulated with or without exosomes. HUVECs migrated to RPE-derived exosomes, and exosomes derived from ARPE-19 cells accelerated HUVEC tube formation. In contrast, Exo-TNF and Exo-CO reduced HUVEC proliferation. Our findings provide insight into the mechanisms underlying the relation between angiogenesis and exosomes derived from RPE cells.

Project description:BACKGROUND:Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells. METHODOLOGY/PRINCIPAL FINDINGS:Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-?1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-?1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38?/? MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38?/? MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-?1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-?1 signaling and by NFAT5 siRNA, respectively. Hyperosmolarity decreased the viability of the cells; this effect was not altered by exogenous bFGF and HB-EGF. Various vegetable polyphenols (luteolin, quercetin, apigenin) inhibited the hyperosmotic expression of bFGF, HB-EGF, and NFAT5 genes. CONCLUSION:Hyperosmolarity induces transcription of bFGF and HB-EGF genes, and secretion of bFGF from RPE cells. This is in part mediated by autocrine/paracrine TGF-?1 and FGF signaling. It is suggested that high intake of dietary salt resulting in osmotic stress may aggravate neovascular retinal diseases via stimulation of the production of angiogenic factors in RPE cells, independent of hypertension.

Dataset Information

Effects of FTMT Expression by Retinal Pigment Epithelial Cells on Features of Angiogenesis.

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Effects of FTMT Expression by Retinal Pigment Epithelial Cells on Features of Angiogenesis.

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