Retinal Pigment Epithelial Cells In Response Research Articles

Blockade of MDM2 with inactive Cas9 prevents epithelial to mesenchymal transition in retinal pigment epithelial cells

Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-β2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-β2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-β2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-β2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-β2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-β2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-β2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-β2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.

Oxidative stress induces mitochondrial dysfunction and autophagy in ARPE‐19 cells

AbstractPurposeOxidative stress plays an important role in the pathogenesis of age‐related macular degeneration (AMD), but the exact mechanism of the influence of the stress is not completely clear. The stress can affect both neural retina and retinal pigment epithelium (RPE) cells The purpose of our study was to evaluate changes in some mitochondrial properties and autophagy in the response of RPE cells to oxidative stress.MethodsRetinal pigment epithelium ARPE‐19 cells were challenged with 200 mM H2O2 for 2 h. Mitochondrial ROS (mtROS) were assessed by the MitoSOX probe, mitochondrial membrane potential (MMP) with JC‐1, the activity of the complex I of the mitochondrial respiratory chain with MitoCheck. To assess autophagy cells were transfected with the pBABE‐puro mCherry‐EGFP‐LC3B plasmid and autophagic flux was evaluated with confocal fluorescent microscopy; expression of the LC3‐I and LC3‐II proteins were quantified by immunoblotting.ResultsHydrogen peroxide induced an increase in mtROS and decrease in MMP in ARPE‐19 cells as well as it inhibited the activity of the complex I. Furthermore, an increase in the LC3‐II/LC3‐I ratio and an increased number of autophagolysosomes was observed in ARPE‐19 cells challenged with hydrogen peroxide.ConclusionOxidative stress induces detrimental changes in mitochondria of RPE cells that can respond to these changes by increased autophagy, confirming that mitochondrial homeostasis and autophagy may be important in AMD pathogenesis.This work was supported by National Science Centre, Poland grant number 2017/27/B/NZ3/00872.

Lipopolysaccharide-Induced Autophagy Mediates Retinal Pigment Epithelium Cells Survival. Modulation by the Phospholipase D Pathway.

Inflammation and oxidative stress are common factors involved in the pathogenesis of retinal diseases, such as aged-related macular degeneration (AMD) and diabetic retinopathy (DR). Autophagy is a catabolic process essential to cell survival in response to stress. This process is highly active in retinal pigment epithelium (RPE) cells. Our previous findings demonstrated that lipopolysaccharide (LPS) induces an inflammatory response of RPE cells that implies classical phospholipases D (PLD1 and 2) activation, cyclooxygenase-2 (COX-2) expression, prostaglandin E2 (PGE2) production and reduced cell viability. In this work, we studied the autophagic process and its modulation by the PLD pathway in D407 and ARPE-19 RPE cells exposed to LPS. LPS (10 μg/ml or 25 μg/ml) exposure for 24 h increased light chain 3B-II (LC3B-II) content (an autophagy marker) and LC3B-positive punctate structures in both RPE cell lines studied. Next, the drug bafilomycin A1 (BAF, 50 nM) was used to block the autophagic flux. In cells pre-incubated with BAF, LC3B-II and sequestosome 1 (SQSTM1/p62) levels and autophagosome-like structures were increased by LPS, demonstrating that the inflammatory injury increases the autophagic process in RPE cells. To study the role of the PLD pathway, cells were pre-incubated for 1 h with selective PLD1 (VU0359595) or PLD2 (VU0285655-1) inhibitors prior to LPS addition. Under control condition, LC3B-positive punctate structures were increased in cells pre-incubated with PLD2 inhibitor while with PLD1 inhibitor were increased in cells exposed to LPS. MTT reduction assays showed that early autophagy inhibitors, 3-methyladenin (3-MA) or LY294002, enhanced the loss in cell viability induced by LPS exposure for 48 h. On the contrary, the inhibition of PLD1 and PLD2 prevented the loss in cell viability induced by LPS. In conclusion, our results show that even though LPS treatment promotes an inflammatory response in RPE cells, it also triggers the activation of the autophagic process which in turn may serve as a protective mechanism for the cells. In addition, we demonstrate that the PLD pathway modulates the autophagic process in RPE cells. Our findings contribute to the knowledge of the molecular basis of retinal inflammatory and degenerative diseases and open new avenues for potential therapeutic exploration.

Retinal Pigment Epithelial Cells Control Early Mycobacterium tuberculosis Infection via Interferon Signaling.

Mycobacterium tuberculosis (Mtb) bacilli have been found in retinal pigment epithelial (RPE) cells from uveitis patients without signs of systemic tuberculosis (TB) infection. RPE cells are important for ocular immune privilege and uveitis development. To address a potential role for Mtb-infected RPE cells in the development of uveitis, we delineated the response to Mtb infection in human RPE cells and primary human macrophages, the main target cell of Mtb. Primary human RPE cells, the human RPE cell line ARPE-19, and monocyte-derived proinflammatory M1 and anti-inflammatory M2 macrophages were infected with DsRed-expressing Mtb strain H37Rv. Infection rates and clearance were addressed along with RNA sequencing analysis, a confirmation analysis by dual-color reverse-transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA) and cytokine secretion. RPE cells robustly controlled intracellular outgrowth of Mtb early after infection. The response in RPE cells to control Mtb survival was dominated by interferon (IFN) signaling and further characterized by prominent regulation of cell death/survival-associated genes and low-level production of Th1-associated cytokines. In contrast, macrophages engaged a plethora of responses including IFN signaling and communication between innate and adaptive immune cells to induce granuloma formation. Together, our data demonstrate that RPE cells display a strong response to Mtb infection that appears, however, incomplete in comparison to the macrophage response to Mtb. The RPE response might reflect a balance between mechanisms aimed at Mtb eradication and mechanisms that limit retinal inflammation.

Activation of the ERK1/2-MAPK Signaling Pathway by Complement Serum in UV-POS-Pretreated ARPE-19 Cells

Background: Retinal pigment epithelial (RPE) cells undergo functional changes upon complement stimulation, which play a role in the pathogenesis of age-related macular degeneration (AMD). These effects are in part enhanced by pretreating ARPE-19 cells with UV-irradiated photoreceptor outer segments (UV-POS) in vitro. The aim of this study was to investigate the effects of human complement serum (HCS) treatment on p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2 [ERK1/2]) activation in ARPE-19 cells pretreated with UV-POS. Methods: UV-POS-pretreated ARPE-19 cells were stimulated with 5% HCS or heat-inactivated HCS (HI-HCS) as a control. Pro tein expression of phosphorylated (activated) ERK1/2, total ERK1/2, Bax, and Bcl-2 was analyzed by Western blotting. Cell culture supernatants were analyzed for IL-6, IL-8, MCP-1, and VEGF by enzyme-linked immunosorbent assay (ELISA). Furthermore, extra- and intracellular reactive oxygen species (ROS) were determined. Results: The amount of phosphorylated ERK1/2 was increased in UV-POS-pretreated ARPE-19 cells, especially in combination with HCS stimulation, compared to non-pretreated ARPE-19 cells incubated with HCS alone or HI-HCS. The same observation was made for Bax and Bcl-2 expression. Furthermore, an increase in extra- and intracellular ROS was detected in UV-POS-pretreated ARPE-19 cells. The ELISA data showed that the production of IL-6, IL-8, and MCP-1 tended to increase in response to HCS in both UV-POS-pretreated and non-pretreated ARPE-19 cells. Conclusions: Our data imply that ERK1/2 activation in ARPE-19 cells may represent a response mechanism to cellular and oxidative stress, associated with apoptosis-regulating factors such as Bax and Bcl-2, which might play a role in AMD, while ERK1/2 seems not to represent the crucial signaling pathway mediating the functional changes in RPE cells in response to complement stimulation.

Autophagy activated by SIRT6 regulates Aβ induced inflammatory response in RPEs

Age-associated dysfunction of retinal pigment epithelial cells (RPEs) is considered to be the initial trigger of retinal diseases such as age-related macular degeneration. Although autophagy is upregulated in RPEs during the course of aging, little is known about how autophagy is regulated and its functional role in RPEs. In this study, we found that expression of Sirtuin 6 (SIRT6) and autophagic markers are upregulated in RPEs of aged mice where subretinal deposition of amyloid-β is accumulated and in amyloid-β stimulated RPEs. In addition, gain and loss-of-function studies confirmed the positive role of SIRT6 in regulating autophagy. Interesting, inhibition of autophagy attenuates amyloid-β stimulated inflammatory response in RPEs. Collectively, our findings uncover the autophagy modulated by SIRT6 may be a proinflammatory mechanism for amyloid-β induced RPE dysfunction.

Long non‑coding RNA associated‑competing endogenous RNAs are induced by clusterin in retinal pigment epithelial cells.

Age related macular degeneration is one of the most common causes of vision loss in the elderly. Long noncoding RNAs (lncRNAs) serve important roles in regulating gene expression by acting as competing endogenous RNAs (ceRNAs). However, the roles of specific lncRNAs and their associated ceRNA function induced by clusterin in cultured retinal pigment epithelial (RPE) cells remain to be fully elucidated. Based on high throughput sequencing data from RPE cells treated with or without clusterin, the present study identified differentially expressed mRNAs, lncRNAs and microRNAs (miRNAs). A lncRNA‑mRNA‑microRNA (miRNA) network (ceRNA network) was subsequently constructed based on the bioinformatic database miRanda and miRNA targets database miRTarBase. These results demonstrated the expression pattern of several lncRNAs, and a clear clusterin‑associated ceRNA network in RPE cells, which included 75 lncRNAs and 32 miRNAs in RPE cells induced by clusterin. Collectively, the present study uncovered and characterized via bioinformatics the global properties of the ceRNA network in human RPE cells in response to clusterin. These results may aid in the elucidation of the molecular mechanisms of clusterin in age‑related macular degeneration.

Homocysteine mediates transcriptional changes of the inflammatory pathway signature genes in human retinal pigment epithelial cells.

To test whether homocysteine (Hcy) can influence the transcriptional profile, we hypothesized that Hcy can lead to the induction of proinflammatory molecules in the retinal cells of aging people. An unbiased in vitro inflammatory pathway focused study was designed employing retinal pigment epithelial (RPE) cell line, ARPE-19. Cells were cultured in the presence or absence of Hcy to capture target genes' expression profile. Three different concentrations of Hcy were added in the culture medium of confluent monolayers. cRNAs were made from the isolated total RNAs and the labeled cRNA probes were hybridized to microarrays specific for human disease pathway inflammatory cytokines, chemokines and their receptor gene micro-array panels as per manufacture's recommendations. Two Hcy up-regulated molecules: IL6 and CEBPB were further validated via Western blot analysis. Hcy's effect on ARPE-19 cellular morphology and genomic DNA integrity were also evaluated. Gene microarray analyses of RPE cells in response to Hcy treatment revealed alterations in the expressions of several inflammatory gene transcripts such as CCL5, CEBPB, IL13RA2, IL15RA, IL6, IL8 and CXCL3 that were up-regulated. The transcripts for C3, CCL2, IL11RA and IL18 genes exhibited down-regulation. The IL6 and CEBPB expressions were subsequently validated at the protein levels. Treatment of the retinal cells with increasing Hcy concentration influenced their density in culture however their morphology and DNA integrity remained unaffected. These findings suggest that Hcy can potentially mediate the expression of chemokines, cytokines and interleukins receptors in the retinal cells without having any debilitating effects on their morphology and the genomic DNA integrity.

Activation of liver X receptor α protects amyloid β1-40 induced inflammatory and senescent responses in human retinal pigment epithelial cells.

To investigate whether activation of the liver X receptors (LXRs) inhibits amyloid β1-40 (Aβ1-40) induced inflammatory and senescent responses in human retinal pigment epithelial (RPE) cells. Confluent cultures of human primary RPE and ARPE-19 cells pretreated with 5μΜ of TO901317 (TO90), a synthetic agonist of LXR, or vehicle were incubated with 1μΜ of Aβ1-40 or Aβ40-1. The optimum concentrations of Aβ1-40 and TO90 were determined by cell viability assay. Pro-inflammatory cytokines IL-6, IL-8, MCP-1 were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Expression and localization of an aging protein p16INK4a (p16) were analyzed by western blotting and immunofluorescence. Expressions of LXRs and one of their target genes ATP-binding cassette transporter A1 (ABCA1) were examined by real-time PCR and western blotting. Phosphorylated transcription inhibition factor-κB-α (p-IκB-α) was assessed by western blotting. A negative linear relationship between the Aβ1-40 concentration and the cell viability was evident, indicating Aβ1-40 decreased ARPE-19 cell viability in a dose-dependent manner. Aβ1-40 enhanced the expression of IL-6, IL-8, MCP-1 as well as p16 in both RPE cell lines at both mRNA and protein levels, whereas TO90 counteracted the detrimental effects. TO90 upregulated the expression of LXRα and its target gene ABCA1, but it did not affect the expression of LXRβ. Meanwhile, TO90 inhibited the phosphorylation of IκB-α mediated by Aβ1-40 stimulation. Activation of the LXRα-ABCA1 axis may alleviate Aβ1-40 induced inflammatory and senescent responses in RPE cells. The beneficial effect appears associated with the inhibition of the NF-κB signaling pathway.

Osmotic induction of placental growth factor in retinal pigment epithelial cells in vitro: contribution of NFAT5 activity.

One risk factor of neovascular age-related macular degeneration is systemic hypertension; hypertension is mainly caused by extracellular hyperosmolarity after consumption of dietary salt. In retinal pigment epithelial (RPE) cells, high extracellular osmolarity induces vascular endothelial growth factor (VEGF)-A (Hollborn et al. in Mol Vis 21:360-377, 2015). The aim of the present study was to determine whether extracellular hyperosmolarity and chemical hypoxia trigger the expression of further VEGF family members including placental growth factor (PlGF) in human RPE cells. Hyperosmotic media were made up by addition of 100mM NaCl or sucrose. Chemical hypoxia was induced by CoCl2. Gene expression was quantified by real-time RT-PCR, and secretion of PlGF-2 was investigated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) was depleted using siRNA. Extracellular hyperosmolarity triggered expression of VEGF-A, VEGF-D, and PlGF genes, and secretion of PlGF-2. Hypoosmolarity decreased PlGF gene expression. Hypoxia induced expression of VEGF-A, VEGF-B, VEGF-D, and PlGF genes. Extracellular hyperosmolarity and hypoxia produced additive PlGF gene expression. Both hyperosmolarity and hypoxia induced expression of KDR and FLT-4 receptor genes, while hyperosmolarity caused neuropilin-2 and hypoxia neuropilin-1 gene expression. The hyperosmotic, but not the hypoxic, PlGF gene expression was in part mediated by NFAT5. The expression of PlGF in RPE cells depends on the extracellular osmolarity. The data suggest that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in the hypoxic retina via transcriptional activation of various VEGF family member genes.

TGF-β1 induced transdifferentiation of rpe cells is mediated by TAK1.

Background and AimProliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells.MethodologyARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis.ResultsThis study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent.Conclusions/SignificanceThis work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment.

Different Effects of Thrombin on VEGF Secretion, Proliferation, and Permeability in Polarized and Non-polarized Retinal Pigment Epithelial Cells

We investigated the effect of thrombin on the secretion of vascular endothelial growth factor (VEGF), on cellular proliferation, and on the integrity of the barrier function of polarized retinal pigment epithelial (RPE) cells. In addition, we compared the responses of polarized to that of non-polarized RPE cells. Porcine polarized RPE cells were established using Transwell membranes. The polarization of the RPE cells was determined by their high transepithelial electrical resistance (TER > 200 Ω cm2) and by their differential secretion of VEGF (basal direction >apical direction by 2.5×). RPE cells were incubated with thrombin (5–20 U/ml) for 24 h. The concentration of VEGF in the culture medium was measured by enzyme-linked immunosorbent assay, and the TER was measured. Cellular proliferation was assessed by Ki-67 immunostaining. The area of laser-induced choroidal naovascularization (CNV) was measured in rat eyes and compare to that of controls with or without thrombin. Our results showed that thrombin significantly increased VEGF secretion both in polarized and non-polarized RPE cells in a dose-dependent way. Thrombin did not significantly affect the TER or the expression of tight-junctional proteins in polarized RPE cells, but decreased it in non-polarized RPE cells by inducing intercellular gaps. Ki-67-positive cells were observed in non-polarized RPE cells but not in polarized RPE cells as controls. After thrombin exposure, the number of Ki-67-positive cells increased significantly in non-polarized RPE cells but not in polarized RPE cells. The area of CNV was larger in thrombin-injected eye than control eyes. Although thrombin increased VEGF secretion regardless of cell polarity, its effects on proliferation and barrier integrity were dependent upon cell polarity. Cell polarization is an important factor for determining the response of RPE cells to thrombin, and the different responsive patterns to thrombin upon cell polarity might explain the complicated pathology of such diseases as age-related macular degeneration.

Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.

Inflammatory cytokines decrease viability and alter ganglioside profile in retinal pigment epithelium cells

AbstractPurpose Early stages of Age related Macular Degeneration (AMD) are characterized by dysfunction and degeneration of the retinal pigment epithelium (RPE) cells, which participate in the death of the overlying photoreceptors ultimately leading to loss of vision. Gangliosides (GG) make a wide and heterogeneous family of sialic‐acid‐containing glycosphingolipids, composed of a sugar chain branched on a ceramide. They are major components of cellular membranes, particularly abundant in the brain and nervous tissue, including retina. While their developmental and neuroprotective actions have been demonstrated, their precise role in retina’s function and its pathologies is still poorly understood. The present study aimed to investigate the role of GG in the response of RPE cells to inflammation, which is known as one of the pathophysiological features of AMD.Methods Cultured human RPE cells (ARPE19) were exposed to an inflammatory cytokine mixture (ICM): TNF‐α, IL‐1β and IFN‐g for 72 hours. Cell viability was assessed and GG were analyzed by Liquid Chromatography coupled with tandem mass spectrometry.Results ICM had deleterious effects on ARPE19 viability: cell number decreased by half between control and treated conditions. GM3 appeared to be the main GG class present in ARPE19 cells. Interestingly, ICM exposure was associated with modifications in the GM3 profile: relative amounts d18:1/16:0 species increased whereas those of d18:1/24:1 species decreased.Conclusion Our observations suggest that GG might be implicated in ARPE19 cell response to inflammatory cytokines, although the precise biological role of the change in fatty acid profile of GM3 still needs to be clarified.

TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.

Lutein and zeaxanthin supplementation reduces photooxidative damage and modulates the expression of inflammation-related genes in retinal pigment epithelial cells

Oxidative damage and inflammation are related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that insufficient dietary lutein and zeaxanthin intake or lower serum zeaxanthin levels are associated with increased risk for AMD. The objective of this work is to test the protective effects of lutein and zeaxanthin against photooxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of inflammation-related genes. To mimic lipofuscin-mediated photooxidation in vivo, we used ARPE-19 cells that accumulated A2E, a lipofuscin fluorophore and photosensitizer, as a model system to investigate the effects of lutein and zeaxanthin supplementation. The data show that supplementation with lutein or zeaxanthin in the medium resulted in accumulation of lutein or zeaxanthin in the RPE cells. The concentrations of lutein and zeaxanthin in the cells were 2- to 14-fold of that detected in the medium, indicating that ARPE-19 cells actively take up lutein or zeaxanthin. As compared with untreated cells, exposure of A2E-containing RPE to blue light resulted in a 40–60% decrease in proteasome activity, a 50–80% decrease in expression of CFH and MCP-1, and an∼20-fold increase in expression of IL-8. The photooxidation-induced changes in expression of MCP-1, IL-8, and CFH were similar to those caused by chemical inhibition of the proteasome, suggesting that inactivation of the proteasome is involved in the photooxidation-induced alteration in expression of these inflammation-related genes. Incubation of the A2E-containing RPE with lutein or zeaxanthin prior to blue light exposure significantly attenuated the photooxidation-induced inactivation of the proteasome and photooxidation-induced changes in expression of MCP-1, IL-8, and CFH. Together, these data indicate that lutein or zeaxanthin modulates inflammatory responses in cultured RPE in response to photooxidation. Protecting the proteasome from oxidative inactivation appears to be one of the mechanisms by which lutein and zeaxanthin modulate the inflammatory response. Similar mechanisms may explain salutary effects of lutein and zeaxanthin in reducing the risk for AMD.

Klebsiella pneumoniae induces an inflammatory response in human retinal-pigmented epithelial cells

PurposeThe retinal pigment epithelium (RPE) is a barrier to Klebsiella pneumoniae infection in endogenous endophthalmitis. Nevertheless, the inflammatory response of RPE cells upon interaction with this pathogen has not been studied. Here we tested the hypothesis that K. pneumoniae induces an inflammatory response in human retinal epithelial cells. MethodsIn this study we set out to investigate the effects of whole K. pneumoniae and of its lipopolysaccharide on RPE cells in vitro using bacterial invasion and cytotoxicity assays, fluorescent microscopy and ELISA. For that, we utilized K. pneumoniae strain ATCC 43816 and the continuous human retinal-pigmented epithelial cell line ARPE-19. ResultsStimulation of ARPE-19 with live K. pneumoniae for 24h induced a 31.5-fold (p=0.0132) increase in IL-6 and 6.5-fold (p=0.0004) increase in MCP-1 levels compared to the non-infected control cells. Purified K. pneumoniae LPS (1μgml−1) also induced cytokine levels, MCP-1 (1.7-fold upregulation; p=0.0006) and IL-6 (1.3-fold upregulation, p=0.065). The tested K. pneumoniae strain ATCC 43816 did not have a significant effect on the viability of ARPE-19 cells (11% decrease, p=0.096) and showed a low ability to invade the cells. ConclusionsBoth whole live K. pneumoniae and K. pneumoniae LPS exert a strong pro-inflammatory effect on retinal pigmented epithelial cells, consistent with clinical manifestations of disease. Bacterial pro-inflammatory effects are not likely related to host cell invasion. This is the first investigation of the interactions of a major endogenous endophthalmitis pathogen, K. pneumoniae with human retinal pigmented epithelial cells.

Effect of hypoxia on expressions of stromal cell-derived factor-1 and integrin-linked kinase in retinal pigment epithelium cell in vitro

Background Hypoxia is a crucial factor of neovascularization.Many researches found that stromal cell-derived factor-1 (SDF-1) and integrin-linked kinase (ILK) play an important role in the neovascular disease.However,effect of SDF-1 and ILK in eye neovascular disease is below understood.Objective The aim of this study was to investigate the effect of hypoxia on the expressions of SDF-1 and ILK in cultured retinal pigment epithelium(RPE) cells in vitro.Methods RPE tissue was isolated from 4-week-old C57BL/6 mouse and was digested and cultured in DMEM/F12 with 10％ fetal bovine serum (FBS).The cells with 80％ confluence were collected and passaged.The third generation of cells were identified with cytokeratin 18 (CK18) antibody by immunochemistry.The cells were inoculated at the density of 5×104 cells/ml to free-serum DMEM/F12 for 24 hours and then were cultured in regular medium in the normoxic control group.RPE cells were cultured for 1 hour and 3,6,12,24,48,72 hours with 200 μmol/L CoCl2 in the hypoxia group.Reverse transcription-PCR(RT-PCR) was used to evaluate the expressing change of SDF-1 mRNA and ILK mRNA in RPE cells,and Western blot was used to assay the expressing change of SDF-1 protein and ILK protein in RPE cells in different time points.The detected outcomes were represented as the ratio of target gene A value/β-actin A value.Results Cultured cells showed the polygon in shape with the black pigment granules in cytoplasm.Over 90％ cells were positive response for CK18.Expressions of the SDF-1 mRNA and ILK mRNA were increased in different time points after CoCl2 co-cultured(SDF-1 mRNA:F=281.875,P=0.000 ;ILK mRNA: F=187.566,P=0.000),with the highest expressing value in hypoxia at 12 hours.No significant change in the expression of SDF-1 mRNA and protein was found 1 hour after CoCl2 co-cultured,but expressions of SDF-1 mRNA and ILK mRNA were significantly higher in 3,6,12,24,48 and 72 hours than the normoxic control group(P＜0.01).The expressions of SDF-1 protein and ILK protein were gradually ascended with the time increase of CoCl2 co-culture,showing a significant difference among different time points(SDF-1: F=44.719,P =0.000 ; ILK: F =144.481,P =0.000),and the up-regulation of SDF-1 protein and ILK protein expression was seen mainly in 3,6,12,24,48 and 72 hours after CoCl2 co-cultured in comparison with the normoxic control group (P＜0.01).Conclusions SDF-1 and ILK are involved in the hypoxic response of RPE cells and may play a potential role in ischemic/hypoxic retinopathy. Key words: Stromal cell-derived factor-1 ; Integrin-linked kinase; Retinal pigment epithelium cell; CoCl2 ; Hypoxia

Inhibition of high glucose-induced VEGF and ICAM-1 expression in human retinal pigment epithelium cells by targeting ILK with small interference RNA

The aim of this study was to investigate the change of Integrin-linked kinase (ILK) expression of human retinal pigment epithelium (RPE) cells in response to high glucose, and the effect of targeting ILK with small interference RNA (siRNA) on the high glucose-induced expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1). The ILK mRNA and protein expression in human RPE cells were analyzed with RT-PCR and western blot after exposure to 5.5, 30, 40, 50 mM glucose, or 5.5 mM glucose+45.5 mM mannitol for 48 h. The expression of VEGF and ICAM-1 was also determined. Cells were treated with ILK siRNA, to determine the effect of ILK on VEGF and ICAM-1 expression following treatment with high glucose. High concentrations of glucose significantly up-regulated ILK mRNA and protein expression, and the ILK expression increased along with the glucose concentration. The changes of VEGF and ICAM-1 expression were similar to that of ILK expression. Knocking down ILK gene expression with siRNA inhibited the elevation of VEGF and ICAM-1 induced by high glucose treatment. These results suggested that ILK was involved in the response of RPE cells to high glucose and may therefore play a role in the pathogenesis of diabetic ophthalmology.

Methylglyoxal‐induced imbalance in the ratio of vascular endothelial growth factor to angiopoietin 2 secreted by retinal pigment epithelial cells leads to endothelial dysfunction

Progressive microvascular complications are a main feature of diabetes and are associated with impairment of the angiogenic response. Methylglyoxal (MGO) has been implicated in the molecular events that lead to endothelial dysfunction in diabetes. In this study, we hypothesize that increased levels of MGO disrupt the ratio of vascular endothelial growth factor (VEGF) to angiopoietin 2 (Ang 2) secreted by retinal pigment epithelial (RPE) cells, which provides a key destabilizing signal that leads to apoptosis and decreased proliferation of retinal endothelial cells. Indeed, we show that MGO increases the levels of Ang 2 and dramatically decreases the levels of VEGF secreted by RPE cells in response to hypoxia. Downregulation of VEGF is likely to be related to decreased hypoxia-inducible factor-1alpha (HIF-1alpha) protein levels and HIF-1 transcriptional activity. Data further show that MGO-induced imbalance in the VEGF/Ang II ratio significantly changes the levels of BAX and Bcl-2 in endothelial cells. Moreover, this imbalance is accompanied by an increase in the activity of caspase-3 and decreased proliferation of endothelial cells. Data obtained in cell culture systems are consistent with observations in retinas of diabetic animals, where increased availability of MGO is associated with changes in distribution and levels of HIF-1alpha, VEGF and Ang 2 and increased microvascular permeability. In conclusion, the MGO-induced imbalance in the VEGF/Ang 2 ratio secreted by retinal epithelial cells activates apoptosis and decreases proliferation of retinal endothelial cells, which are likely to contribute to endothelial dysfunction in diabetic retinopathy.