Extracellular Soluble Membranes from Retinal Pigment Epithelial Cells Mediate Apoptosis in Macrophages.
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ABSTRACT:
SUBMITTER: Sanjiv N
PROVIDER: S-EPMC8153131 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Shiga toxins (Stxs) produced by Shiga toxin-producing bacteria Shigella dysenteriae serotype 1 and select serotypes of Escherichia coli are the most potent known virulence factors in the pathogenesis of hemorrhagic colitis progressing to potentially fatal systemic complications such as acute renal failure, blindness and neurological abnormalities. Although numerous studies have defined apoptotic responses to Shiga toxin type 1 (Stx1) or Shiga toxin type 2 (Stx2) in a variety of cell types, the potential significance of Stx-induced apoptosis of photoreceptor and pigmented cells of the eye following intoxication is unknown. We explored the use of immortalized human retinal pigment epithelial (RPE) cells as an in vitro model of Stx-induced retinal damage. To the best of our knowledge, this study is the first report that intoxication of RPE cells with Stxs activates both apoptotic cell death signaling and the endoplasmic reticulum (ER) stress response. Using live-cell imaging analysis, fluorescently labeled Stx1 or Stx2 were internalized and routed to the RPE cell endoplasmic reticulum. RPE cells were significantly sensitive to wild type Stxs by 72 h, while the cells survived challenge with enzymatically deficient mutant toxins (Stx1A- or Stx2A-). Upon exposure to purified Stxs, RPE cells showed activation of a caspase-dependent apoptotic program involving a reduction of mitochondrial transmembrane potential (Δψm), increased activation of ER stress sensors IRE1, PERK and ATF6, and overexpression CHOP and DR5. Finally, we demonstrated that treatment of RPE cells with Stxs resulted in the activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), suggesting that the ribotoxic stress response may be triggered. Collectively, these data support the involvement of Stx-induced apoptosis in ocular complications of intoxication. The evaluation of apoptotic responses to Stxs by cells isolated from multiple organs may reveal unique functional patterns of the cytotoxic actions of these toxins in the systemic complications that follow ingestion of toxin-producing bacteria.
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Project description:We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC-MS/MS based proteomics. EV-associated transforming growth factor beta 1 (TGFβ1) protein was measured by enzyme-linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT- or R345W-EVs. The role of EV-bound TGFβ was determined by pre-incubation of EVs with a pan-TGFβ blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W- EVs, while EMT drivers were enriched. Additionally, R345W-EVs had higher concentrations of TGFβ1 compared to control. Critically, treatment with R345W-EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV-increased cell migration was significantly inhibited by pre-incubation of EVs with pan-TGFβ-neutralising antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFβ dependent manner. These results suggest that EV-bound TGFβ plays a critical role in the induction of EMT in RPE cells.
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Project description:In the eye, the retinal pigment epithelium (RPE) adheres to a complex protein matrix known as Bruch's membrane (BrM). The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM) resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM) was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin) or the outer layers (collagen VI). ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.
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Project description:Macular fibrosis is a vital obstacle of vision acuity improvement of age-related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL-2) on epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor ?2 (TGF-?2) expression in retinal pigment epithelial (RPE) cells. 10 ?g/L IL-2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker ?-smooth muscle actin (?-SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL-1), TGF-?2, and the activation of the JAK/STAT3 and NF-?B signaling pathway. Furthermore, JAK/STAT3 and NF-?B signaling pathways were specifically blocked by WP1066 or BAY11-7082, respectively, and the expression of ?-SMA, COL-1, Fn and TGF-?2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL-2 with or without WP1066 or BAY11-7082. After induction of IL-2, the expressions of Fn, COL-1, TGF-?2 protein were significantly increased, and this effect was correlated with IL-2 treatment duration, while ?-SMA protein expression did not change significantly. Both WP1066 and BAY11-7082 could effectively downregulate the expression of Fn, COL-1 and TGF-?2 induced by IL-2. What's more, both NF-?B and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF-?B inhibitor BAY 11-7082 could obviously decrease RPE cells migration capability induced by IL-2. IL-2 promotes cell migration, ECM synthesis and TGF-?2 expression in RPE cells via JAK/STAT3 and NF-?B signaling pathways, which may play an important role in proliferative vitreoretinopathy.
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Project description:αB-Crystallin is a protein chaperone with anti-apoptotic and anti-inflammatory activity that is apically secreted in exosomes by polarized human retinal pigment epithelium. A 20 amino acid mini-peptide derived from residues 73-92 of αB-crystallin protects human retinal pigment epithelial (RPE) cells from oxidative stress, a process involved in the progression of age-related macular degeneration (AMD). Unfortunately, due to its small size, its development as a therapeutic requires a robust controlled release system. To achieve this goal, the αB-crystallin peptide was re-engineered into a protein polymer nanoparticle/macromolecule with the purpose of increasing the hydrodynamic radius/molecular weight and enhancing potency via multivalency or an extended retention time. The peptide was recombinantly fused with two high molecular weight (~40kDa) protein polymers inspired by human tropoelastin. These elastin-like polypeptides (ELPs) include the following: (i) a soluble peptide called S96 and (ii) a diblock copolymer called SI that assembles multivalent nanoparticles at physiological temperature. Fusion proteins, cryS96 and crySI, were found to reduce aggregation of alcohol dehydrogenase and insulin, which demonstrates that ELP fusion did not diminish chaperone activity. Next their interaction with RPE cells was evaluated under oxidative stress. Unexpectedly, H2O2-induced stress dramatically enhanced cellular uptake and nuclear localization of both cryS96 and crySI ELPs. Accompanying uptake, both fusion proteins protected RPE cells from apoptosis, as indicated by reduced caspase 3 activation and TUNEL staining. This study demonstrates the in vitro feasibility of modulating the hydrodynamic radius for small peptide chaperones by seamless fusion with protein polymers; furthermore, they may have therapeutic applications in diseases associated with oxidative stress, such as AMD.
| S-EPMC4222838 | biostudies-literature
Project description:AimTo identify the effect and regulatory mechanism of amyloid β (Aβ) protein on retinal pigment epithelial (RPE) cells in cell proliferation and apoptosis, and clarify Aβ role in the pathogenesis of age-related macular degeneration (AMD).MethodsThe model of Aβ25-35 protein cytotoxicity in RPE cell was successfully established to investigate the effect of Aβ protein on RPE cells in vitro. Based on Aβ protein, the specific inhibitors (HY-50682 or BAY11-7082) or activating agent (lipopolysaccharide) was used to analyze the regulatory mechanism of Aβ protein to RPE cells on cell proliferation and apoptosis by flow cytometry, real-time polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay and dual-luciferase reporter gene assay.ResultsThe number of RPE cells, treated with Aβ25-35 from 0.3 to 60 µmol/L, significantly reduce (P<0.01), and had the dose-dependent effect. Aβ protein 60 µmol/L inhibits the G1/S phase transition (P<0.01) and down-regulated cyclin E mRNA level (P<0.01). Similarly, Aβ25-35 induced a significant increase of cell apoptosis, accompanied by the significantly higher level of activated caspase 3 protein. Furthermore, nuclear factor-kappaB (NF-κB) activity and phosphorylated Iκ-Ba level would significantly lower in treated RPE cells. Using specific inhibitors or activating agent based on the Aβ, the cell numbers, NF-κB activity, phosphorylated Iκ-Ba level, receptor for advanced glycation endproducts (RAGE) gene expression levels, cyclin E mRNA level and activated caspase 3 level had accordingly changed by different methods, confirming that RAGE/NF-κB signaling pathway involved in the regulation of Aβ protein on RPE cell apoptosis and proliferation.ConclusionAβ protein inhibits cell proliferation and activates apoptosis via inactivation of the RAGE/NF-κB signaling pathway in RPE cell.
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Project description:OBJECTIVE:This paper applied a transcriptomic approach to investigate the mechanisms of adriamycin (ADR) in treating proliferative vitreoretinopathy (PVR) using ARPE-19 cells. METHODS:The growth inhibitory effects of ADR on ARPE-19 cells were assessed by sulforhodamine B (SRB) assay and propidium iodide (PI) staining using flow cytometry. The differentially expressed genes between ADR-treated ARPE-19 cells and normal ARPE-19 cells and the signaling pathways involved were investigated by microarray analysis. Mitochondrial function was detected by JC-1 staining using flow cytometry and the Bcl-2/Bax protein family. The phosphorylated histone H2AX (?-H2AX), phosphorylated checkpoint kinase 1 (p-CHK1), and phosphorylated checkpoint kinase 2 (p-CHK2) were assessed to detect DNA damage and repair. RESULTS:ADR could significantly inhibit ARPE-19 cell proliferation and induce caspase-dependent apoptosis in vitro. In total, 4479 differentially expressed genes were found, and gene ontology items and the p53 signaling pathway were enriched. A protein-protein interaction analysis indicated that the TP53 protein molecules regulated by ADR were related to DNA damage and oxidative stress. ADR reduced mitochondrial membrane potential and the Bcl-2/Bax ratio. p53-knockdown restored the activation of c-caspase-3 activity induced by ADR by regulating Bax expression, and it inhibited ADR-induced ARPE-19 cell apoptosis. Finally, the levels of the ?-H2AX, p-CHK1, and p-CHK2 proteins were up-regulated after ADR exposure. CONCLUSIONS:The mechanism of ARPE-19 cell death induced by ADR may be caspase-dependent apoptosis, and it may be regulated by the p53-dependent mitochondrial dysfunction, activating the p53 signaling pathway through DNA damage.
| S-EPMC6305253 | biostudies-literature
Project description:Diabetic retinopathy (DR) is a common complication of diabetes mellitus and results in acquired blindness among working-age adults. It has been demonstrated that high glucose (HG)-induced oxidative stress and cell apoptosis in retinal pigment epithelial (RPE) cells are major factors for the pathogenesis of DR. Sauchinone, one of the active lignan isolated from Saururus chinensis, was reported to possess anti-oxidant and anti-apoptosis effects. In the present study, we investigated the effects of sauchinone on HG-induced oxidative stress and apoptosis in ARPE-19 cells. Our results proved that sauchinone improved the cell viability of HG-induced ARPE-19 cells. Moreover, sauchinone treatment caused a decrease in intracellular reactive oxygen species (ROS) generation and an increase in the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). Besides, flow cytometry showed that the apoptotic rate in sauchinone-treated ARPE-19 cells obviously decreased as compared in the HG-treated cells. Western blot indicated that sauchinone treatment caused a significant decrease in bax expression and increase in bcl-2 expression in HG-treated ARPE-19 cells. Sauchinone treatment enhanced the HG-caused induction of p-Akt, nuclear factor erythroid 2-related factor (Nrf2), and heme oxygenase-1 (HO-1) expressions in ARPE-19 cells. However, the inhibitor of Akt, LY294002, reversed the effects of sauchinone on cell viability, oxidative stress, and cell apoptosis in HG-treated ARPE-19 cells. These findings suggested that sauchinone treatment prevented HG-induced oxidative stress and apoptosis via regulating the Akt/Nrf2/HO-1 pathway in HG-induced RPE cells. These findings suggested that sauchinone might be a therapeutic agent for the treatment and prevention of DR.
| S-EPMC9064550 | biostudies-literature
Project description:Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance.
| S-EPMC5394430 | biostudies-literature
Project description:PurposeTo investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells.MethodsDot-blot and hydroxymethylated DNA immunoprecipitation sequencing were applied to evaluate the impact of ascorbate on DNA hydroxymethylation in ARPE-19 cells. RNA sequencing (RNA-seq) was carried out to analyze the transcriptome. Quantitative RT-PCR and ELISA were conducted to examine the transcription and secretion of VEGF from cultured cells. Primary human fetal RPE cells and RPE-J cells were used to verify the effect of ascorbate on VEGF expression. ELISA was used to measure VEGF in the vitreous humor of Gulo-/- mice, which, like humans, cannot synthesize ascorbate de novo.ResultsTreatment with ascorbate (50 μM) promoted 5-hydroxymethycytosine (5hmC) generation and changed the genome-wide profiles of 5hmC in ARPE-19 cells. Ascorbate also caused a dramatic shift in the transcriptome-3186 differential transcripts, of which 69.3% are correlated with altered 5hmC in promoters or gene bodies. One of the most downregulated genes was VEGFA, which encodes the VEGF protein. The suppression of VEGF by ascorbate is independent of hypoxia-inducible factor 1-alpha (HIF-1α) but correlates with increased 5hmC in the gene body. The decreased transcription and secretion of VEGF by ascorbate were verified in primary human fetal RPE cells. Furthermore, adding ascorbate in the diet for Gulo-/- mice resulted in decreased levels of VEGF in the RPE/choroid and vitreous humor.ConclusionsAscorbate inhibits VEGF expression in RPE cells likely via DNA hydroxymethylation. Thus, ascorbate could be implicated in the prevention or treatment of diseases such as age-related macular degeneration (AMD).
| S-EPMC6049987 | biostudies-literature