Epithelial-mesenchymal Transition (EMT) and the Effect of Atorvastatin on it in ARPE-19 cells.

Proliferative vitreoretinopathy (PVR) develops after an unsuccessful or complicated recovery from rhegmatogenous retinal detachment (RRD) surgery. Intraocular scar formation with the contribution of epithelial-mesenchymal transition (EMT) in RPE cells is prominent in the pathology of PVR. In the present study, the EMT process was experimentally induced in human retinal pigment epithelium (RPE; ARPE-19) cells, and the effect of atorvastatin on the process was studied. The mRNA and protein levels of mesenchymal markers actin alpha 2 (ACTA2) / alpha-smooth muscle actin (α-SMA) and fibronectin (FN), and epithelial markers occludin (OCLN) and zonula occludens-1 (ZO-1) were measured using quantitative real-time PCR (qRT-PCR) and western blot methods, respectively. In addition, α-SMA and FN were visualized using immunofluorescence staining. Cells were photographed under a phase contrast light microscope. Changes in the functionality of cells following the EMT process were studied using the IncuCyte scratch wound cell migration assay and the collagen cell invasion assay with confocal microscopy. The induction of EMT in ARPE-19 cells increased the expression of mesenchymal markers ACTA2/α-SMA and fibronectin and reduced the expression of epithelial marker OCLN both at mRNA and protein levels. The mRNA levels of ZO-1 were lower after EMT, as well. Increased levels of α-SMA and FN were confirmed by immunofluorescence staining. Atorvastatin further increased the mRNA levels of mesenchymal markers ACTA2 and FN as well as the protein levels of α-SMA and reduced the mRNA levels of epithelial markers OCLN and ZO-1 under the EMT process. EMT promoted wound closure and cell invasion into the 3D collagen matrix when compared to untreated control cells. These data present cellular changes upon the induction of the EMT process in ARPE-19 cells and the propensity of atorvastatin to complement the effect. More studies are needed to confirm the exact influence of the EMT process and atorvastatin treatment on the PVR development after RRD surgery.

Cis-urocanic acid improves cell viability and suppresses inflammasome activation in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a common eye disease among the elderly, which can result in impaired vision and irreversible loss of vision. The majority of patients suffer from the dry (also known as the atrophic) form of the disease, which is completely lacking an effective treatment. In the present study, we evaluated the potential of cis-urocanic acid (cis-UCA) to protect human ARPE-19 cells from cell damage and inflammasome activation induced by UVB light. Urocanic acid is a molecule normally present in human epidermis. Its cis-form has recently been found to alleviate UVB-induced inflammasome activation in human corneal epithelial cells. Here, we observed that cis-UCA is well-tolerated also by human retinal pigment epithelial (RPE) cells at a concentration of 100 µg/ml. Moreover, cis-UCA was cytoprotective and efficiently diminished the levels of mature IL-1ß, IL-18, and cleaved caspase-1 in UVB-irradiated ARPE-19 cells. Interestingly, cis-UCA also reduced DNA damage, whereas its effect against ROS production was negligible. Collectively, cis-UCA protected ARPE-19 cells from UVB-induced phototoxicity and inflammasome activation. This study indicates that due to its beneficial properties of preserving cell viability and preventing inflammation, cis-UCA has potential in drug development of chronic ocular diseases, such as AMD.

Antimycin A-induced mitochondrial dysfunction regulates inflammasome signaling in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a severe retinal eye disease where dysfunctional mitochondria and damaged mitochondrial DNA in retinal pigment epithelium (RPE) have been demonstrated to underlie the pathogenesis of this devastating disease. In the present study, we aimed to examine whether damaged mitochondria induce inflammasome activation in human RPE cells. Therefore, ARPE-19 cells were primed with IL-1α and exposed to the mitochondrial electron transport chain complex III inhibitor, antimycin A. We found that antimycin A-induced mitochondrial dysfunction caused caspase-1-dependent inflammasome activation and subsequent production of mature IL-1ß and IL-18 in human RPE cells. AIM2 and NLRP3 appeared to be the responsible inflammasome receptors upon antimycin A-induced mitochondrial damage. We aimed at verifying our findings using hESC-RPE cells but antimycin A was absorbed by melanin. Therefore, results were repeated on D407 RPE cell cultures. Antimycin A-induced mitochondrial and NADPH oxidase-dependent ROS production occurred upstream of inflammasome activation, whereas K+ efflux was not required for inflammasome activation in antimycin A-treated human RPE cells. Collectively, our data emphasize that dysfunctional mitochondria regulate the assembly of inflammasome multiprotein complexes in the human RPE cells. The present study associates AIM2 with the pathogenesis of AMD.

TAS-116, a Well-Tolerated Hsp90 Inhibitor, Prevents the Activation of the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells.

Chronic inflammation has been associated with several chronic diseases, such as age-related macular degeneration (AMD). The NLRP3 inflammasome is a central proinflammatory signaling complex that triggers caspase-1 activation leading to the maturation of IL-1ß. We have previously shown that the inhibition of the chaperone protein, Hsp90, prevents NLRP3 activation in human retinal pigment epithelial (RPE) cells; these are cells which play a central role in the pathogenesis of AMD. In that study, we used a well-known Hsp90 inhibitor geldanamycin, but it cannot be used as a therapy due to its adverse effects, including ocular toxicity. Here, we have tested the effects of a novel Hsp90 inhibitor, TAS-116, on NLRP3 activation using geldanamycin as a reference compound. Using our existing protocol, inflammasome activation was induced in IL-1α-primed ARPE-19 cells with the proteasome and autophagy inhibitors MG-132 and bafilomycin A1, respectively. Intracellular caspase-1 activity was determined using a commercial caspase-1 activity kit and the FLICA assay. The levels of IL-1ß were measured from cell culture medium samples by ELISA. Cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and lactate dehydrogenase (LDH) measurements. Our findings show that TAS-116 could prevent the activation of caspase-1, subsequently reducing the release of mature IL-1ß. TAS-116 has a better in vitro therapeutic index than geldanamycin. In summary, TAS-116 appears to be a well-tolerated Hsp90 inhibitor, with the capability to prevent the activation of the NLRP3 inflammasome in human RPE cells.

Effects of Resvega on Inflammasome Activation in Conjunction with Dysfunctional Intracellular Clearance in Retinal Pigment Epithelial (RPE) Cells.

Age-related macular degeneration (AMD) is an eye disease in which retinal pigment epithelium (RPE) cells play a crucial role in maintaining retinal homeostasis and photoreceptors' functionality. During disease progression, there is increased inflammation with nucleotide-binding domain, leucine-rich repeat, and Pyrin domain 3 (NLRP3) inflammasome activation, oxidative stress, and impaired autophagy in RPE cells. Previously, we have shown that the dietary supplement Resvega reduces reactive oxygen species (ROS) production and induces autophagy in RPE cells. Here, we investigated the ability of Resvega to prevent NLRP3 inflammasome activation with impaired protein clearance in human RPE cells. Cell viability was measured using the lactate dehydrogenase (LDH) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Enzyme-linked immunosorbent assays (ELISA) were utilized to determine the secretion of cytokines, NLRP3, and vascular endothelial growth factor (VEGF). Caspase-1 activity was measured with a fluorescent labeled inhibitor of caspase-1 (FLICA; FAM-YVAD-FMK) and detected microscopically. Resvega improved the cell membrane integrity, which was evident as reduced LDH leakage from cells. In addition, the caspase-1 activity and NLRP3 release were reduced, as was the secretion of two inflammatory cytokines, interleukin (IL)-1ß and IL-8, in IL-1α-primed ARPE-19 cells. According to our results, Resvega can potentially reduce NLRP3 inflammasome-mediated inflammation in RPE cells with impaired protein clearance.

UV-B-Induced Inflammasome Activation Can Be Prevented by Cis-Urocanic Acid in Human Corneal Epithelial Cells.

Purpose: The cornea is continually exposed to highly energetic solar UV-B (280-320 nm). Our aim was to investigate whether UV-B triggers the activation of NLRP3 inflammasomes and the production of IL-1ß and/or IL-18 in human corneal epithelial (HCE) cells. Additionally, we studied the capability of cis-urocanic acid (cis-UCA) to prevent inflammasome activation or alleviate inflammation through other signaling pathways. Methods: HCE-2 cell line and primary HCE cells were primed using lipopolysaccharide or TNF-α. Thereafter, cells were exposed to UV-B before or after the addition of cis-UCA or caspase-1 inhibitor. Caspase-1 activity was measured from cell lysates by an enzymatic assay. IL-1ß, IL-18, IL-6, IL-8, and NLRP3 levels were detected using the ELISA method from cell culture media. Additionally, intracellular NLRP3 levels were determined by the Western blot technique, and cytotoxicity was measured by the LDH assay. Results: UV-B exposure significantly increased caspase-1 activity in TNF-α-primed HCE cells. This result was consistent with the concurrently induced IL-1ß secretion. Both caspase-1 activity and release of IL-1ß were reduced by cis-UCA. Additionally, UV-B stimulated the caspase-1-independent production of IL-18, an effect also reduced by cis-UCA. Cis-UCA decreased the release of IL-6, IL-8, and LDH in a time-dependent manner when administered to HCE-2 cells after UV-B exposure. Conclusions: Our findings demonstrate that UV-B activates inflammasomes in HCE cells. Cis-UCA can prevent the secretion of IL-1ß and IL-18 and therapeutically reduces the levels of IL-6, IL-8, and LDH in UV-B-stressed HCE cells.

Only IL-1ß release is inflammasome-dependent upon ultraviolet B irradiation although IL-18 is also secreted.

DNA damage accumulates in aged postmitotic retinal pigment epithelium (RPE) cells, a phenomenon associated with the development of age-related macular degeneration. In this study, we have experimentally induced DNA damage by ultraviolet B (UVB) irradiation in interleukin-1α (IL-1α)-primed ARPE-19 cells and examined inflammasome-mediated signaling. To reveal the mechanisms of inflammasome activation, cells were additionally exposed to high levels of extracellular potassium chloride, n-acetyl-cysteine, or mitochondria-targeted antioxidant MitoTEMPO, prior to UVB irradiation. Levels of interleukin-18 (IL-18) and IL-1ß mRNAs were detected with qRT-PCR and secreted amounts of IL-1ß, IL-18, and caspase-1 were measured with ELISA. The role of nucleotide-binding domain and leucine-rich repeat pyrin containing protein 3 (NLRP3) in UVB-induced inflammasome activation was verified by using the NLRP3-specific siRNA. Reactive oxygen species (ROS) levels were measured immediately after UVB exposure using the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2 DCFDA) indicator, the levels of cyclobutane pyrimidine dimers were assayed by cell-based ELISA, and the extracellular levels of adenosine triphosphate (ATP) determined using a commercial bioluminescence assay. We found that pro-IL-18 was constitutively expressed by ARPE-19 cells, whereas the expression of pro-IL-1ß was inducible by IL-1α priming. UVB induced the release of mature IL-18 and IL-1ß but NLRP3 contributed only to the secretion of IL-1ß. At the mechanistic level, the release of IL-1ß was regulated by K+ efflux, whereas the secretion of IL-18 was dependent on ROS production. As well as K+ efflux, the cells released ATP following UVB exposure. Collectively, our data suggest that UVB clearly stimulates the secretion of mature IL-18 as a result of ROS induction, and this response is associated with DNA damage. Moreover, in human RPE cells, K+ efflux mediates the UVB-activated NLRP3 inflammasome signaling, leading to the processing of IL-1ß.

SQSTM1/p62 regulates the production of IL-8 and MCP-1 in IL-1ß-stimulated human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a complex eye disease in which decline in autophagy leads to the accumulation of sequestosome 1/p62 (SQSTM1/p62)-labeled waste material inside the retinal pigment epithelial (RPE) cells, and the condition results in activation of the inflammasome signaling and IL-1ß secretion. Here, we have studied the role of SQSTM1/p62 in the production of IL-6, IL-8, and MCP-1 in the presence or absence of IL-1ß. SQSTM1/p62 was either overexpressed or silenced in ARPE-19 cells, which were then exposed to IL-1ß. Alternatively, bafilomycin A was used to demonstrate the functional decline of autophagy with increased SQSTM1/p62 levels. The protein concentration of SQSTM1/p62 was measured using the western blot technique, and interleukin levels were determined by ELISA. In IL-1ß-loaded RPE cells, SQSTM1/p62 depletion and overexpression increased the production of MCP-1 and IL-8, respectively. Neither knock-down nor overexpression of SQSTM1/p62 induced the release of IL-6. Our data suggest that SQSTM1/p62 is a significant factor in inflammatory responses, especially following the inflammasome activation.

Oxidative Stress is the Principal Contributor to Inflammasome Activation in Retinal Pigment Epithelium Cells with Defunct Proteasomes and Autophagy.

BACKGROUND/AIMS: Previously, we demonstrated that blockade of the intracellular clearance systems in human retinal pigment epithelial (RPE) cells by MG-132 and bafilomycin A1 (BafA) induces NLRP3 inflammasome signaling. Here, we have explored the activation mechanisms behind this process. NLRP3 is an intracellular receptor detecting factors ranging from the endogenous alarmins and adenosine triphosphate (ATP) to ultraviolet radiation and solid particles. Due to the plethora of triggers, the activation of NLRP3 is often indirect and can be mediated through several alternative pathways. Potassium efflux, lysosomal rupture, and oxidative stress are currently the main mechanisms associated with many activators. METHODS: NLRP3 inflammasomes were activated in human RPE cells by blocking proteasomes and autophagy using MG-132 and bafilomycin A1 (BafA), respectively. P2X7 inhibitor A740003, potassium chloride (KCl), and glyburide, or N-acetyl-L-cysteine (NAC), ammonium pyrrolidinedithiocarbamate (APDC), diphenyleneiodonium chloride (DPI), and mito-TEMPO were added to cell cultures in order to study the role of potassium efflux and oxidative stress, respectively. IL-1ß was measured using the ELISA method. ATP levels and cathepsin B activity were examined using commercial kits, and ROS levels using the fluorescent dye 2´,7´-dichlorodihydrofluorescein diacetate (DCFDA). RESULTS: Elevated extracellular potassium prevented the priming factor IL-1α from inducing the production of reactive oxygen species (ROS). It also prevented IL-1ß release after exposure of primed cells to MG-132 and BafA. Inflammasome activation increased extracellular ATP levels, which did not appear to trigger significant potassium efflux. The activity of the lysosomal enzyme, cathepsin B, was reduced by MG-132 and BafA, suggesting that cathepsin B was not playing any role in this phenomenon. Instead, MG-132 triggered ROS production already 30 min after exposure, but treatment with antioxidants blocking NADPH oxidase and mitochondria-derived ROS significantly prevented IL-1ß release after this activating signal. CONCLUSION: Our data suggest that oxidative stress strongly contributes to the NLRP3 inflammasome activation upon dysfunctional cellular clearance. Clarification of inflammasome activation mechanisms provides novel options for alleviating pathological inflammation present in aggregation diseases, such as age-related macular disease (AMD) and Alzheimer's disease.

Hsp90 inhibition as a means to inhibit activation of the NLRP3 inflammasome.

Once activated, the intracellular receptor NLRP3 assembles an inflammasome protein complex that facilitates the caspase-1-mediated maturation of IL-1ß and IL-18. Inactive NLRP3 is guarded by a protein complex containing Hsp90. In response to stress stimuli, Hsp90 is released, and NLRP3 can be activated to promote inflammation. In this study, we blocked Hsp90 with geldanamycin and studied the fate of NLRP3 in human retinal pigment epithelial (RPE) cells. RPE cells play a central role in the development of age-related macular degeneration (AMD), a progressive eye disease causing severe vision loss in the elderly. IL-1α-primed ARPE-19 cells, human embryonal stem cell (hESC)-derived RPE cells, and primary human RPE cells were exposed to MG-132 and bafilomycin A to activate NLRP3 via the inhibition of proteasomes and autophagy, respectively. Additionally, RPE cells were treated with geldanamycin at different time points and the levels of NLRP3 and IL-1ß were determined. Caspase-1 activity was measured using a commercial assay. Geldanamycin prevented the activation of the inflammasome in human RPE cells. NLRP3 released from its protective complex became degraded by autophagy or secreted from the cells. Controlled destruction of NLRP3 is a potential way to regulate the inflammation associated with chronic diseases, such as AMD.

Hypoxia and inflammation in the release of VEGF and interleukins from human retinal pigment epithelial cells.

PURPOSE: Retinal diseases are closely associated with both decreased oxygenation and increased inflammation. It is not known if hypoxia-induced vascular endothelial growth factor (VEGF) expression in the retina itself evokes inflammation, or whether inflammation is a prerequisite for the development of neovascularization. METHODS: Human ARPE-19 cell line and primary human retinal pigment epithelium (RPE) cells were used. ARPE-19 cells were kept either under normoxic (24 h or 48 h) or hypoxic conditions (1% O2, 24 h). Part of the cells were re-oxygenated (24 h). Some ARPE-19 cells were additionally pre-treated with bacterial lipopolysaccharide (LPS). The levels of IL-6, IL-8, IL-1ß, and IL-18 were determined from medium samples by an enzyme-linked immunosorbent assay (ELISA) method. Primary human RPE cells were exposed to hypoxia for 24 h, and the subsequent release of IL-6 and IL-8 was measured with ELISA. VEGF secretion from ARPE-19 cells was determined up to 24 h. RESULTS: Hypoxia induced significant (P < 0.01) increases in the levels of both IL-6 and IL-8 in ARPE-19 cells, and LPS pre-treatment further enhanced these responses. Hypoxia exposure did not affect the IL-1ß or IL-18 release irrespective of LPS pre-treatment. If primary RPE cells were incubated for 4 h in hypoxic conditions, IL-6 and IL-8 concentrations were increased by 7 and 8-fold respectively. Hypoxia increased the VEGF secretion from ARPE-19 cells in a similar manner with or without pre-treatment with LPS. CONCLUSIONS: Hypoxia causes an inflammatory reaction in RPE cells that is potentiated by pre-treatment with the Toll-like receptor-activating agent, LPS. The secretion of VEGF from these cells is regulated directly by hypoxia and is not mediated by inflammation.

NLRP3 inflammasome activation is associated with proliferative diabetic retinopathy.

PURPOSE: Innate immunity and dysregulation of inflammatory processes play a role in vascular diseases like atherosclerosis or diabetes. Nucleotide-binding domain and Leucine-rich repeat Receptor containing a Pyrin domain 3 (NLRP3) inflammasomes are pro-inflammatory signalling complexes that were found in 2002. In addition to pathogens and other extracellular threats, they can be activated by various endogenous danger signals. The purpose of this study was to find out whether NLRP3 activation occurs in patients with sight-threatening forms of diabetic retinopathy (DR). METHODS: Inflammasome components NLRP3 and caspase-1, inflammasome-related pro-inflammatory cytokines IL-1ß and IL-18, vascular endothelial growth factor (VEGF), acute-phase cytokines TNF-α and IL-6, as well as adaptive immunity-related cytokine interferon gamma (IFN-γ) were measured from the vitreous samples of 15 non-proliferative diabetic retinopathy (non-PDR) and 23 proliferative diabetic retinopathy (PDR) patients using the enzyme-linked immunosorbent assay (ELISA) method. The adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) was determined using the Western blot technique. RESULTS: Inflammasome components were present in the vitreous of DR patients. Along with VEGF, the levels of caspase-1 and IL-18 were significantly increased, especially in PDR eyes. Interestingly, clearly higher levels of NLRP3 were found in the PDR eyes with tractional retinal detachment (TRD) than from PDR eyes with fully attached retina. There were no significant differences in the amounts of IL-1ß, TNF-α, IL-6, and IFN-γ that were detectable in the vitreous of both non-PDR and PDR patients. CONCLUSION: Our results suggest that NLRP3 inflammasome activation can be associated especially with the pathogenesis of PDR. The lack of differences in TNF-α, IL-6, and IFN-γ also alludes that acute inflammation or T-cell-mediated responses do not dominate in PDR pathogenesis.

Two dietary polyphenols, fisetin and luteolin, reduce inflammation but augment DNA damage-induced toxicity in human RPE cells.

Plant-derived polyphenols are known to possess anti-inflammatory and antioxidant effects. In recent years, several studies have investigated their potential benefits for treating chronic diseases associated with prolonged inflammation and excessive oxidative stress, such as age-related macular degeneration (AMD). Previously, two polyphenols, fisetin and luteolin, have been reported to increase the survival of retinal pigment epithelial (RPE) cells suffering from oxidative stress as well as decreasing inflammation but the benefits of polyphenol therapy seem to depend on the model system used. Our aim was to analyze the effects of fisetin and luteolin on inflammation and cellular viability in a model of nonoxidative DNA damage-induced cell death in human RPE (hRPE) cells. Pretreatment of ARPE-19 or primary hRPE cells with the polyphenols augmented etoposide-induced cell death as measured by the lactate dehydrogenase and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. However, the treatment was able to reduce the release of two proinflammatory cytokines, IL-6 and IL-8, which were determined by enzyme-linked Immunosorbent assay. Analyses of caspase 3 activity, p53 acetylation and SIRT1 protein levels revealed the apoptotic nature of etoposide-evoked cell death and that fisetin and luteolin augmented the etoposide-induced acetylation of p53 and decreased SIRT1 levels. Taken together, our findings suggest that the cytoprotective effects of fisetin and luteolin depend on the stressor they need to combat, whereas their anti-inflammatory potential is sustained over a variety of model systems. Careful consideration of disease pathways will be necessary before fisetin or luteolin can be recommended as therapeutic agents for inflammatory diseases in general and specifically AMD.

Fisetin and luteolin protect human retinal pigment epithelial cells from oxidative stress-induced cell death and regulate inflammation.

Degeneration of retinal pigment epithelial (RPE) cells is a clinical hallmark of age-related macular degeneration (AMD), the leading cause of blindness among aged people in the Western world. Both inflammation and oxidative stress are known to play vital roles in the development of this disease. Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation. We also compare the growth and reactivity of human ARPE-19 cells in serum-free and serum-containing conditions. The absence of serum in the culture medium did not prevent ARPE-19 cells from reaching full confluency but caused an increased sensitivity to oxidative stress-induced cell death. Both fisetin and luteolin protected ARPE-19 cells from oxidative stress-induced cell death. They also significantly decreased the release of pro-inflammatory cytokines into the culture medium. The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1. The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD.

Quercetin alleviates 4-hydroxynonenal-induced cytotoxicity and inflammation in ARPE-19 cells.

Retinal pigment epithelium (RPE) plays the principal role in age-related macular degeneration (AMD), a progressive eye disease with no cure and limited therapeutical options. In the pathogenesis of AMD, degeneration of RPE cells by multiple factors including increased oxidative stress and chronic inflammation precedes the irreversible loss of photoreceptors and central vision. Here, we report that the plant-derived polyphenol, quercetin, increases viability and decreases inflammation in stressed human ARPE-19 cells after exposure to the lipid peroxidation end product 4-hydroxynonenal (HNE). Several previous studies have been conducted using the direct oxidant H2O2 but we preferred HNE since natural characteristics predispose RPE cells to the type of oxidative damage evoked by lipid peroxidation. Quercetin improved cell membrane integrity and mitochondrial function as assessed in LDH and MTT tests. Decreased production of proinflammatory mediators IL-6, IL-8, and MCP-1 were indicated at the RNA level by qPCR and at the protein level by the ELISA technique. In addition, we probed the signaling behind the effects and observed that p38 and ERK MAPK pathways, and CREB signaling are regulated by quercetin in ARPE-19 cells. In conclusion, our present data suggests that HNE is highly toxic to serum-starved ARPE-19 cells but quercetin is able to reverse these adverse effects even when administered after an oxidative insult.

Decline in cellular clearance systems induces inflammasome signaling in human ARPE-19 cells.

Retinal pigment epithelium (RPE) plays a major role in the maintenance of photoreceptors, and degeneration of RPE results in the development of age-related macular degeneration (AMD). Accumulation of intracellular protein aggregates, increased oxidative stress, and chronic inflammation are all factors damaging the functionality of aged RPE cells. Here, we report that inhibition of proteasomal degradation with MG-132 and autophagy with bafilomycin A1 resulted in the release of IL-1ß but not that of IL-18 in human ARPE-19 cells. NLRP3 receptor became upregulated, and caspase-1, the functional component of an inflammasome complex, was activated. In addition to accumulating intracellular protein aggregates, inhibition of degradation systems induced oxidative stress which was demonstrated by elevated amounts of intracellular 4-hydroxynonenal (HNE)-protein adducts. Along with IL-1ß, exposure to MG-132 and bafilomycin A1 resulted in the secretion of IL-8. A low concentration (1pg/ml) of IL-1ß was capable of triggering significant IL-8 production which also became attenuated by treatment with a specific caspase-1 inhibitor. These results suggest that decline in intracellular degradation systems results not only in increased amounts of intracellular protein aggregates and oxidative stress but also in the activation of NLRP3 inflammasomes, arisen as a result of elevated production of biologically active IL-1ß.

Cationorm shows good tolerability on human HCE-2 corneal epithelial cell cultures.

Preservatives have been for a long time known to cause detrimental effects on ocular surface. Cationorm, a preservative-free compound with electrostatic properties is a novel way to solve the problems encountered with traditional benzalkonium chloride (BAK)-containing eye drops. The aim of this study was to evaluate tolerability of the preservative-free cationic emulsion Cationorm in vitro on corneal epithelial cells. The human corneal epithelial cell (HCE-2) culture line was used to study cellular morphology, cytotoxicity and inflammatory responses after Cationorm diluted 1/10 exposure for 5, 15 and 30 min. Exposures to Systane diluted 1/10 with polyquaternium-1/polidronium chloride 0.001% as preservative, BAK 0.001% or C16 (0.0002%) and normal cell culture medium served as positive and negative references. Cell viability was determined by measuring the release of lactate dehydrogenase (LDH) and mitochondrial dehydrogenase activity was evaluated using 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The possible induction of apoptosis was analyzed by measuring the activity of caspase-3, and Cell Counting Kit-8 (CCK-8) was used to evaluate the number of viable cells after the exposure to test compounds. Furthermore, the tendency of the test compounds to produce inflammatory reaction was determined by analyzing the production of proinflammatory cytokines IL-6 and IL-8, and DNA binding of the p65 subunit of transcription factor NF-κB was measured from cell lysates. HCE-2 cells showed no morphological changes after the exposure to Cationorm, but in cells exposed to BAK, clear cytoplasm vacuolization and loose cell-cell contacts were observed in transmission (TEM) or scanning (SEM) electron microscopic analyses. Cell viability, as measured with the release of LDH, indicated a time dependent increase in LDH expression after exposure to all test compounds but especially with BAK. Moreover, Cationorm and BAK time-dependently decreased the mitochondrial metabolism to 73% with Cationorm and 53% with BAK from that of the control cells after 30 min exposure in MTT assay. BAK was the only test compound having clear adverse effects on the cell number and metabolism in CCK-8 assay. The activity of caspase-3 did not show significant differences between the groups. Inflammatory response after exposure to Cationorm was significantly lower than after exposure to BAK. There were no significant differences in NF-κB activity between the groups. Diluted Cationorm and Systane with polyquaternium-1/polidronium chloride 0.001% showed good tolerability on HCE-2 cells and thereby provide a clear improvement when compared to BAK-containing eye drop formulations.