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: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:AimTo identify the effect and regulatory mechanism of amyloid ? (A?) protein on retinal pigment epithelial (RPE) cells in cell proliferation and apoptosis, and clarify A? role in the pathogenesis of age-related macular degeneration (AMD).MethodsThe model of A?25-35 protein cytotoxicity in RPE cell was successfully established to investigate the effect of A? protein on RPE cells in vitro. Based on A? protein, the specific inhibitors (HY-50682 or BAY11-7082) or activating agent (lipopolysaccharide) was used to analyze the regulatory mechanism of A? 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 A?25-35 from 0.3 to 60 µmol/L, significantly reduce (P<0.01), and had the dose-dependent effect. A? protein 60 µmol/L inhibits the G1/S phase transition (P<0.01) and down-regulated cyclin E mRNA level (P<0.01). Similarly, A?25-35 induced a significant increase of cell apoptosis, accompanied by the significantly higher level of activated caspase 3 protein. Furthermore, nuclear factor-kappaB (NF-?B) activity and phosphorylated I?-Ba level would significantly lower in treated RPE cells. Using specific inhibitors or activating agent based on the A?, the cell numbers, NF-?B activity, phosphorylated I?-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-?B signaling pathway involved in the regulation of A? protein on RPE cell apoptosis and proliferation.ConclusionA? protein inhibits cell proliferation and activates apoptosis via inactivation of the RAGE/NF-?B signaling pathway in RPE cell.
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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.
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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.
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Project description:Purpose:To investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells. Methods:Dot-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. Results:Treatment with ascorbate (50 ?M) 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-1?) 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. Conclusions:Ascorbate 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).
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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.
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Project description:The accumulation and formation of advanced glycation end products (AGEs) are related to diabetes and age-related disease. Osteomeles schwerinae C. K. Schneid. (Rosaceae, OSSC) is used traditionally for the treatment of various diseases in Asia. Previous studies have shown that OSSC elicits preventive effects in an in vivo model of diabetes. This study was to evaluate the antiapoptotic effects of dried leaves and twigs of OSSC extract and its major compounds in ARPE-19 cells-spontaneously arising human retinal pigment epithelial cells-under diabetic conditions. To examine the effects of an OSSC extract and its active compounds (acetylvitexin, hyperoside and quercitrin) on apoptosis in methylglyoxal (MG, the active precursor in the formation of AGEs)-treated ARPE-19 cells and the mechanism by which these effects occur, apoptosis was measured using flow cytometry analysis. Protein expression levels of phospho-p53 (p-p53), Bax and Bcl-2 were determined by western blot analyses. The OSSC extract inhibited apoptosis in MG-treated ARPE-19 cells in a dose-dependent manner. The major compounds also reduced the rate of apoptosis. Both the extract and major compounds also inhibited the expression of p-p53 and Bax and increased the levels of Bcl-2 that had been previously reduced by MG treatment. The OSSC extract (0.1 ?g/mL) and its major compounds (0.01 ?M) attenuated apoptosis in ARPE-19 cells under toxic diabetic conditions by downregulating of expression of p-p53 and Bax. OSSC may serve as an alternative therapy to retard the development of diabetic retinopathy.
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Project description:Proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) are characterized by the development of epi-retinal membranes which may exert a tractional force on retina. A lot of inflammatory growth factors may disturb the local ocular cells such as retinal pigment epithelial (RPE) cells, causing them to migrate and proliferate in the vitreous cavity and ultimately forming the PVR membrane. In this study, the signal pathways mediating cell migration of RPE induced by growth factors were investigated. Hepatocyte growth factor (HGF), epidermal growth factor (EGF) or heparin-binding epidermal growth factor (HB-EGF) induced a greater extent of migration of RPE50 and ARPE19 cells, compared with other growth factors. According to inhibitor studies, migration of RPE cells induced by each growth factor was mediated by protein kinase C (PKC) and ERK (MAPK). Moreover, HGF coupled with EGF or HB-EGF had synergistic effects on cell migration and enhanced activation of PKC and ERK, which were attributed to cross activation of growth factor receptors by heterogeneous ligands. Furthermore, using the shRNA technique, PKC? was found to be the most important PKC isozyme involved. Finally, vitreous fluids from PVR and PDR patients with high concentration of HGF may induce RPE cell migration in PKC?- and ERK- dependent manner. In conclusion, migration of RPE cells can be synergistically induced by HGF coupled with HB-EGF or EGF, which were mediated by enhanced PKC? activation and ERK phosphorylation.
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