Kir4.2 Potassium Channels in Retinal Pigment Epithelial Cells In Vitro: Contribution to Cell Viability and Proliferation, and Down-Regulation by Vascular Endothelial Growth Factor.

Dedifferentiation and proliferation of retinal pigment epithelial (RPE) cells are characteristics of retinal diseases. Dedifferentiation is likely associated with changes of inwardly rectifying potassium (Kir) channels. The roles of Kir4.2 channels in viability, and proliferation of cultured RPE cells were investigated. Gene expression levels were determined using qRT-PCR. RPE cells expressed Kir2.1, 2.2, 2.4, 3.2, 4.1, 4.2, 6.1, and 7.1 mRNA. Kir4.2 protein was verified by immunocytochemistry and Western blotting. Kir4.2 mRNA in cultured cells was upregulated by hypoxia (hypoxia mimetic CoCl2 or 0.2% O2) and extracellular hyperosmolarity (addition of high NaCl or sucrose). Kir4.2 mRNA was suppressed by vascular endothelial growth factor (VEGF), blood serum, and thrombin whereas platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and transforming growth factor-ß1 (TGF-ß1) increased it. Hyperosmotic Kir4.2 gene expression was mediated by TGF-ß1 receptor signaling while hypoxic gene transcription was dependent on PDGF receptor signaling. VEGF receptor-2 blockade increased Kir4.2 mRNA level under control, hyperosmotic, and hypoxic conditions. SiRNA-mediated knockdown of Kir4.2 decreased the cell viability and proliferation under control and hyperosmotic conditions. Kir4.2 channels play functional roles in maintaining the viability and proliferation of RPE cells. Downregulation of Kir4.2 by VEGF, via activation of VEGF receptor-2 and induction of blood-retinal barrier breakdown, may contribute to decreased viability of RPE cells under pathological conditions.

Hypoxic and osmotic expression of Kir2.1 potassium channels in retinal pigment epithelial cells: Contribution to vascular endothelial growth factor expression.

Retinal pigment epithelial (RPE) cells express different subtypes of inwardly rectifying potassium (Kir) channels. We investigated whether human and rat RPE cells express genes of strongly rectifying Kir2 channels. We also determined the hypoxic and hyperosmotic regulation of Kir2.1 gene expression in cultured human RPE cells and the effects of siRNA-mediated knockdown of Kir2.1 on VEGFA expression, VEGF secretion, proliferation, and viability of the cells. Extracellular hyperosmolarity was induced by addition of NaCl or sucrose. Hypoxia and chemical hypoxia were produced by cell culture in 0.25% O2 and addition of CoCl2, respectively. Gene expression levels were evaluated by real-time RT-PCR. Rat RPE cells contained Kir2.1, Kir2.2, Kir2.3, and Kir2.4 gene transcripts while human RPE cells contained Kir2.1, Kir2.2, and Kir2.4 transcripts. Immunocytochemical data may suggest that Kir2.1 protein in cultured human cells is expressed in both perinuclear and plasma membranes. Kir2.1 gene expression and Kir2.1 protein level in human cells increased under hypoxic and hyperosmotic conditions. The expression of the Kir2.1 gene was mediated in part by diverse intracellular signal transduction pathways and transcription factor activities under both conditions; the hyperosmotic, but not the CoCl2-induced Kir2.1 gene expression was dependent on intracellular calcium signaling. Autocrine/paracrine activation of purinergic receptors contributed to Kir2.1 gene expression under hyperosmotic (P2Y1, P2Y2, P2X7) and CoCl2-induced conditions (P2Y2, P2X7). Exogenous VEGF, TGF-ß1, and blood serum decreased Kir2.1 gene expression. Inhibition of VEGF receptor-2 increased the Kir2.1 gene expression under control conditions and in CoCl2-simulated hypoxia, and decreased it under high NaCl conditions. Knockdown of Kir2.1 by siRNA inhibited the CoCl2-induced and hyperosmotic transcription of the VEGFA gene and caused a delayed decrease of the constitutive VEGFA gene expression while VEGF protein secretion was not altered. Kir2.1 knockdown stimulated RPE cell proliferation under control and hyperosmotic conditions without affecting cell viability. The data indicate that Kir2.1 channel activity is required for the expression of the VEGFA gene and inhibits the proliferation of RPE cells. Under control and hypoxic conditions, the extracellular VEGF level may regulate the production of VEGF via its inhibitory effect on the Kir2.1 gene transcription; this feedback loop may prevent overproduction of VEGF.

Foveal regeneration after resolution of cystoid macular edema without and with internal limiting membrane detachment: presumed role of glial cells for foveal structure stabilization.

AIM: To document with spectral-domain optical coherence tomography the morphological regeneration of the fovea after resolution of cystoid macular edema (CME) without and with internal limiting membrane (ILM) detachment and to discuss the presumed role of the glial scaffold for foveal structure stabilization. METHODS: A retrospective case series of 38 eyes of 35 patients is described. Of these, 17 eyes of 16 patients displayed foveal regeneration after resolution of CME, and 6 eyes of 6 patients displayed CME with ILM detachment. Eleven eyes of 9 patients displayed other kinds of foveal and retinal disorders associated with ILM detachment. RESULTS: The pattern of edematous cyst distribution, with or without a large cyst in the foveola and preferred location of cysts in the inner nuclear layer or Henle fiber layer (HFL), may vary between different eyes with CME or in one eye during different CME episodes. Large cysts in the foveola may be associated with a tractional elevation of the inner foveal layers and the formation of a foveoschisis in the HFL. Edematous cysts are usually not formed in the ganglion cell layer. Eyes with CME and ILM detachment display a schisis between the detached ILM and nerve fiber layer (NFL) which is traversed by Müller cell trunks. ILM detachment was also found in single eyes with myopic traction maculopathy, macular pucker, full-thickness macular holes, outer lamellar holes, and glaucomatous parapapillary retinoschisis, and in 3 eyes with Müller cell sheen dystrophy (MCSD). As observed in eyes with MCSD, cellophane maculopathy, and macular pucker, respectively, fundus light reflections can be caused by different highly reflective membranes or layers: the thickened and tightened ILM which may or may not be detached from the NFL, the NFL, or idiopathic epiretinal membranes. In eyes with short single or multiple CME episodes, the central fovea regenerated either completely, which included the disappearance of irregularities of the photoreceptor layer lines and the reformation of a fovea externa, or with remaining irregularities of the photoreceptor layer lines. CONCLUSION: The examples of a complete regeneration of the foveal morphology after transient CME show that the fovea may withstand even large tractional deformations and has a conspicuous capacity of structural regeneration as long as no cell degeneration occurs. It is suggested that the regenerative capacity depends on the integrity of the threedimensional glial scaffold for foveal structure stabilization composed of Müller cell and astrocyte processes. The glial scaffold may also maintain the retinal structure after loss of most retinal neurons as in late-stage MCSD.

Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y2 receptor signaling.

Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O2 atmosphere and addition of CoCl2, respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1ß levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O2 induced expression of NLRP3 and pro-IL-1ß genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1ß proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y2 and adenosine A1 receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y2 (but not A1) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y2 receptor signaling.

Osmotic and hypoxic induction of the complement factor C9 in cultured human retinal pigment epithelial cells: Regulation of VEGF and NLRP3 expression.

Purpose: Variants of complement factor genes, hypoxia and oxidative stress of the outer retina, and systemic hypertension affect the risk of age-related macular degeneration. Hypertension often results from the high intake of dietary salt that increases extracellular osmolarity. We determined the effects of extracellular hyperosmolarity, hypoxia, and oxidative stress on the expression of complement genes in cultured (dedifferentiated) human RPE cells and investigated the effects of C9 siRNA and C9 protein on RPE cells. Methods: Hyperosmolarity was induced by adding 100 mM NaCl or sucrose to the culture medium. Hypoxia was induced by culturing cells in 1% O2 or by adding the hypoxia mimetic CoCl2. Oxidative stress was induced by adding H2O2. Gene and protein expression levels were determined with real-time RT-PCR, western blot, and ELISA analyses. The expression of the nuclear factor of activated T cell 5 (NFAT5) and complement factor (C9) was knocked down with siRNA. Results: Extracellular hyperosmolarity, hypoxia, and oxidative stress strongly increased the transcription of the C9 gene, while the expression of the C3, C5, CFH, and CFB genes was moderately altered or not altered at all. Hyperosmolarity also induced a moderate increase in the cytosolic C9 protein level. The hyperosmotic C9 gene expression was reduced by inhibitors of the p38 MAPK, ERK1/2, JNK, and PI3K signal transduction pathways and of the transcription factors STAT3 and NFAT5. The hypoxic C9 gene expression was reduced by a STAT3 inhibitor. The knockdown of C9 with siRNA decreased the hypoxic vascular endothelial growth factor (VEGF) and NLRP3 gene expression, the hypoxic secretion of VEGF, and the hyperosmotic expression of the NLRP3 gene. Exogenous C9 protein inhibited the hyperosmotic expression of the C9 gene, the hypoxic and hyperosmotic VEGF gene expression, and the hyperosmotic expression of the NLRP3 gene. Both C9 siRNA and C9 protein inhibited inflammasome activation under hyperosmotic conditions, as indicated by the decrease in the cytosolic level of mature IL-1ß. Conclusions: The expression of the C9 gene in cultured RPE cells is highly induced by extracellular hyperosmolarity, hypoxia, and oxidative stress. The data may support the assumption that C9 gene expression may stimulate the expression of inflammatory (NLRP3) and angiogenic growth factors (VEGF) in RPE cells. Extracellular C9 protein may attenuate this effect, in part via negative regulation of the C9 mRNA level.

Osmotic regulation of NFAT5 expression in RPE cells: The involvement of purinergic receptor signaling.

PURPOSE: Systemic hypertension is a risk factor for age-related neovascular retinal diseases. The major condition that induces hypertension is the intake of dietary salt (NaCl) resulting in increased extracellular osmolarity. High extracellular NaCl was has been shown to induce angiogenic factor production in RPE cells, in part via the transcriptional activity of nuclear factor of activated T cell 5 (NFAT5). Here, we determined the signaling pathways that mediate the osmotic expression of the NFAT5 gene in RPE cells. METHODS: Cultured human RPE cells were stimulated with high (+100 mM) NaCl. Alterations in gene and protein expression were determined with real-time reverse transcriptase (RT)-PCR and western blot analysis, respectively. RESULTS: NaCl-induced NFAT5 gene expression was fully inhibited by calcium chelation and blockers of inositol triphosphate (IP3) receptors and phospholipases C and A2. Blockers of phospholipases C and A2 also prevented the NaCl-induced increase of the cellular NFAT5 protein level. Inhibitors of multiple intracellular signaling transduction pathways and kinases, including p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), phosphatidylinositol-3 kinase (PI3K), protein kinases A and C, Src tyrosine kinases, and calpains, as well as cyclooxygenase inhibitors, decreased the NaCl-induced expression of the NFAT5 gene. In addition, autocrine purinergic signaling mediated by a release of ATP and a nucleoside transporter-mediated release of adenosine, activation of P2X7, P2Y1, P2Y2, and adenosine A1 receptors, but not adenosine A2A receptors, is required for the full expression of the NFAT5 gene under hyperosmotic conditions. NaCl-induced NFAT5 gene expression is in part dependent on the activity of nuclear factor κB (NF-κB). The NaCl-induced expression of NFAT5 protein was prevented by inhibitors of phospholipases C and A2 and an inhibitor of NF-κB, but it was not prevented by a P2Y1 inhibitor. CONCLUSIONS: The data suggest that in addition to calcium signaling and activation of inflammatory enzymes, autocrine/paracrine purinergic signaling contributes to the stimulatory effect of hyperosmotic stress on the expression of the NFAT5 gene in RPE cells. It is suggested that high intake of dietary salt induces RPE cell responses, which may contribute to age-related retinal diseases.

Intake of dietary salt and drinking water: Implications for the development of age-related macular degeneration.

PURPOSE: Systemic hypertension is a risk factor of age-related retinal diseases such as diabetic retinopathy and age-related macular degeneration. High intake of dietary salt and low intake of water increase extracellular osmolality resulting in hypertension, in particular in salt-sensitive individuals. This review summarizes the present knowledge regarding the impact of salt and water intake on the regulation of blood pressure, retinal function, and the development of age-related retinal diseases. METHODS: A literature search of the Medline database and a summary of recent studies that used human RPE cells. RESULTS: The salt sensitivity of the blood pressure and plasma osmolality increase with age, and body water deficits are common in older individuals. High plasma osmolality has adverse effects in the retina. In RPE cells, high osmolality induces expression and secretion of angiogenic factors, such as vascular endothelial growth factor (VEGF), placental growth factor, and basic fibroblast growth factor, and expression of aquaporin-5, a water channel implicated in transepithelial water transport. The transcriptional activities of hypoxia-inducible factor-1 (HIF-1) and nuclear factor of activated T cell 5 (NFAT5) are critical for the production of VEGF in response to salt-induced osmotic stress. Salt-induced osmotic stress also induces priming of the NLRP3 inflammasome and activates inflammatory enzymes in RPE cells. CONCLUSIONS: Raised plasma osmolality may aggravate age-related retinal diseases by stimulation of local inflammation and angiogenic factor production in the RPE. Alterations in salt and water consumption, and of minerals that stimulate renal salt excretion, may offer nutritional approaches to prevent age-related retinal disorders, in particular in salt-sensitive individuals and individuals who show signs of body dehydration.

P2Y1 Receptor Signaling Contributes to High Salt-Induced Priming of the NLRP3 Inflammasome in Retinal Pigment Epithelial Cells.

BACKGROUND: Systemic hypertension is a risk factor of age-related macular degeneration (AMD), a chronic inflammatory disease. Acute hypertension is caused by increased extracellular osmolarity after intake of dietary salt (NaCl). We determined in cultured human retinal pigment epithelial (RPE) cells whether high extracellular NaCl alters the gene expression of inflammasome-associated proteins, and whether autocrine/paracrine purinergic (P2) receptor signaling contributes to the NaCl-induced NLRP3 gene expression. METHODOLOGY/PRINCIPAL FINDINGS: Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Gene and protein expression levels were determined with real-time RT-PCR and Western blot analysis, respectively. IL-1ß and IL-18 levels were evaluated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. High extracellular NaCl induced NLRP3 and pro-IL-1ß gene expression, while the gene expression of further inflammasome-associated proteins (NLRP1, NLRP2, NLRP6, NLRP7, NLRP12, NLRC4, AIM2, ASC, procaspase-1, pro-IL-18) was not altered or below the detection threshold. The NaCl-induced NLRP3 gene expression was partially dependent on the activities of phospholipase C, IP3 receptors, protein kinase C, the serum and glucocorticoid-regulated kinase, p38 MAPK, ERK1/2, JNK, PI3K, and the transcription factors HIF-1 and NFAT5. Pannexin-dependent ATP release and P2Y1 receptor activation is required for the full induction of NLRP3 gene expression. High NaCl induced a transient increase of the NLRP3 protein level and a moderate NLRP3 inflammasome activation, as indicated by the transient increase of the cytosolic level of mature IL-1ß. High NaCl also induced secretion of IL-18. CONCLUSION: High extracellular NaCl induces priming of the NLRP3 inflammasome in RPE cells, in part via P2Y1 receptor signaling. The inflammasome priming effect of NaCl suggests that high intake of dietary salt may promote local retinal inflammation implicated in the development of AMD.

Osmotic expression of aldose reductase in retinal pigment epithelial cells: involvement of NFAT5.

BACKGROUND: Diabetic retinopathy is associated with osmotic stress resulting from hyperglycemia and intracellular sorbitol accumulation. Systemic hypertension is a risk factor of diabetic retinopathy. High intake of dietary salt increases extracellular osmolarity resulting in systemic hypertension. We determined the effects of extracellular hyperosmolarity, chemical hypoxia, and oxidative stress on the gene expression of enzymes involved in sorbitol production and conversion in cultured human retinal pigment epithelial (RPE) cells. METHODS: Alterations in the expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH) genes were examined with real-time RT-PCR. Protein levels were determined with Western blot analysis. Nuclear factor of activated T cell 5 (NFAT5) was knocked down with siRNA. RESULTS: AR gene expression in RPE cells was increased by high (25 mM) extracellular glucose, CoCl2 (150 µM)-induced chemical hypoxia, H2O2 (20 µM)-induced oxidative stress, and extracellular hyperosmolarity induced by addition of NaCl or sucrose. Extracellular hyperosmolarity (but not hypoxia) also increased AR protein level. SDH gene expression was increased by hypoxia and oxidative stress, but not extracellular hyperosmolarity. Hyperosmolarity and hypoxia did not alter the SDH protein level. The hyperosmotic AR gene expression was dependent on activation of metalloproteinases, autocrine/paracrine TGF-ß signaling, activation of p38 MAPK, ERK1/2, and PI3K signal transduction pathways, and the transcriptional activity of NFAT5. Knockdown of NAFT5 or inhibition of AR decreased the cell viability under hyperosmotic (but not hypoxic) conditions and aggravated the hyperosmotic inhibition of cell proliferation. CONCLUSIONS: The data suggest that sorbitol accumulation in RPE cells occurs under hyperosmotic, but not hypoxic and oxidative stress conditions. NFAT5- and AR-mediated sorbitol accumulation may protect RPE cells under conditions of osmotic stress.

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 100 mM 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.

Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells.

BACKGROUND: Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells. METHODOLOGY/PRINCIPAL FINDINGS: Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-ß1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-ß1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/ß MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/ß MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-ß1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-ß1 signaling and by NFAT5 siRNA, respectively. Hyperosmolarity decreased the viability of the cells; this effect was not altered by exogenous bFGF and HB-EGF. Various vegetable polyphenols (luteolin, quercetin, apigenin) inhibited the hyperosmotic expression of bFGF, HB-EGF, and NFAT5 genes. CONCLUSION: Hyperosmolarity induces transcription of bFGF and HB-EGF genes, and secretion of bFGF from RPE cells. This is in part mediated by autocrine/paracrine TGF-ß1 and FGF signaling. It is suggested that high intake of dietary salt resulting in osmotic stress may aggravate neovascular retinal diseases via stimulation of the production of angiogenic factors in RPE cells, independent of hypertension.

Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA.

PURPOSE: The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting. RESULTS: Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1ß, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes. CONCLUSIONS: The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation.

Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: involvement of NFAT5.

PURPOSE: High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration. Neovascular retinal diseases are associated with edema. Various factors and channels, including vascular endothelial growth factor (VEGF) and aquaporins (AQPs), influence neovascularization and the development of edema. Therefore, we determined whether extracellular hyperosmolarity alters the expression of VEGF and AQPs in cultured human retinal pigment epithelial (RPE) cells. METHODS: Human RPE cells obtained within 48 h of donor death were prepared and cultured. Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. The levels of signaling proteins and nuclear factor of activated T cell 5 (NFAT5) were determined by western blotting. DNA binding of NFAT5 was determined with EMSA. NFAT5 was knocked down with siRNA. RESULTS: Extracellular hyperosmolarity stimulated VEGF gene transcription and the secretion of VEGF protein. Hyperosmolarity also increased the gene expression of AQP5 and AQP8, induced the phosphorylation of p38 MAPK and ERK1/2, increased the expression of HIF-1α and NFAT5, and induced the DNA binding of NFAT5. The hyperosmotic expression of VEGF was dependent on the activation of p38 MAPK, ERK1/2, JNK, PI3K, HIF-1, and NFAT5. The hyperosmotic induction of AQP5 was in part dependent on the activation of p38 MAPK, ERK1/2, NF-κB, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia. CONCLUSIONS: Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells. The data suggest that high salt intake resulting in osmotic stress may aggravate neovascular retinal diseases and edema via the stimulation of VEGF production in RPE. The downregulation of AQP5 under hypoxic conditions may prevent the resolution of edema.

Nonvesicular release of ATP from rat retinal glial (Müller) cells is differentially mediated in response to osmotic stress and glutamate.

Retinal glial (Müller) cells release ATP upon osmotic stress or activation of metabotropic glutamate receptors. ATP inhibits the osmotic Müller cell swelling by activation of P2Y1 receptors. In the present study, we determined the molecular pathways of the ATP release from Müller cells in slices of the rat retina. Administration of the ATP/ADPase apyrase induced a swelling of Müller cells under hypoosmotic conditions, and prevented the swelling-inhibitory effect of glutamate, suggesting that swelling inhibition is mediated by extracellular ATP. A hypoosmotic swelling of Müller cells was also observed in the presence of a blocker of multidrug resistance channels (MK-571), a CFTR inhibitor (glibenclamide), and connexin hemichannel blockers (18-α-glycyrrhetinic acid, 100 µM carbenoxolone). The swelling-inhibitory effect of glutamate was prevented by MK-571, the connexin hemichannel blockers, and a pannexin-1 hemichannel blocker (5 µM carbenoxolone). The p-glycoprotein blocker verapamil had no effect. As revealed by single-cell RT-PCR, subpopulations of Müller cells expressed mRNAs for pannexin-1 and -2, and connexins 30, 30.3, 32, 43, 45, and 46. The data may suggest that rat Müller cells release ATP by multidrug resistance channels, CFTR, and connexin hemichannels in response to osmotic stress, while glutamate induces a release of ATP via multidrug resistance channels, connexin hemichannels, and pannexin-1.

Effects of the vegetable polyphenols epigallocatechin-3-gallate, luteolin, apigenin, myricetin, quercetin, and cyanidin in primary cultures of human retinal pigment epithelial cells.

PURPOSE: Vegetable polyphenols (bioflavonoids) have been suggested to represent promising drugs for treating cancer and retinal diseases. We compared the effects of various bioflavonoids (epigallocatechin-3-gallate [EGCG], luteolin, apigenin, myricetin, quercetin, and cyanidin) on the physiological properties and viability of cultured human retinal pigment epithelial (RPE) cells. METHODS: Human RPE cells were obtained from several donors within 48 h of death. Secretion of vascular endothelial growth factor (VEGF) was determined with enzyme-linked immunosorbent assay. Messenger ribonucleic acid levels were determined with real-time reverse transcription polymerase chain reaction. Cellular proliferation was investigated with a bromodeoxyuridine immunoassay, and chemotaxis was examined with a Boyden chamber assay. The number of viable cells was determined by Trypan Blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation enzyme-linked immunosorbent assay. The phosphorylation level of signaling proteins was revealed by western blotting. RESULTS: With the exception of EGCG, all flavonoids tested decreased dose-dependently the RPE cell proliferation, migration, and secretion of VEGF. EGCG inhibited the secretion of VEGF evoked by CoCl2-induced hypoxia. The gene expression of VEGF was reduced by myricetin at low concentrations and elevated at higher concentrations. Luteolin, apigenin, myricetin, and quercetin induced significant decreases in the cell viability at higher concentration, by triggering cellular necrosis. Cyanidin reduced the rate of RPE cell necrosis. Myricetin caused caspase-3 independent RPE cell necrosis mediated by free radical generation and activation of calpain and phospholipase A2. The myricetin- and quercetin-induced RPE cell necrosis was partially inhibited by necrostatin-1, a blocker of programmed necrosis. Most flavonoids tested diminished the phosphorylation levels of extracellular signal-regulated kinases 1/2 and Akt proteins. CONCLUSIONS: The intake of luteolin, apigenin, myricetin, and quercetin as supplemental cancer therapy or in treating retinal diseases should be accompanied by careful monitoring of the retinal function. The possible beneficial effects of EGCG and cyanidin, which had little effect on RPE cell viability, in treating retinal diseases should be examined in further investigations.

Effects of arteriolar constriction on retinal gene expression and Müller cell responses in a rat model of branch retinal vein occlusion.

BACKGROUND: To investigate the effect of induced arteriolar constriction (AC) on alterations in gene expression of factors implicated in the development of edema in branch retinal vein occlusion (BRVO). METHODS: In Brown-Norway rats, BRVO was induced by laser photocoagulation of the veins in one half of the retina. AC of the afferent arterioles was performed 30 min later. We then determined the expression of Vegfa, Vegfb, Pedf, Kir4.1, Aqp4, Aqp1, Il1ß, and Il6 with real-time polymerase chain reaction (RT-PCR) in the neuroretina and retinal pigment epithelium (RPE) after 1, 3, and 7 days. Immunostaining against GFAP, aquaporin (AQP)-4, and Kir4.1 was performed on days 1 and 3. RESULTS: BRVO resulted in transient upregulation of Vegfa in the neuroretina on day 1. The expressions of Kir4.1, AQP4, and AQP1 were downregulated, and Il1ß and Il6 were strongly upregulated, on days 1 and 3. The retinal distribution of GFAP and AQP4 proteins remained unaltered, while the Kir4.1 protein displayed redistribution from polarized to uniform retinal distribution. AC accelerated the restoration of downregulated Kir4.1, Aqp4, and Aqp1 in the RPE, of Kir4.1 in the neuroretina, and of upregulated Il6 in the neuroretina. AC did not influence the gliotic alterations of Müller cells and the redistribution of the Kir4.1 protein. CONCLUSION: Constriction of the afferent artery in the BRVO region accelerated the restoration of potassium channels and Il6. These alterations may contribute to faster resorption of retinal edema, and may decrease the level of inflammation.

Cytotoxic effects of curcumin in human retinal pigment epithelial cells.

BACKGROUND: Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. CONCLUSION: It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of cancer or in the treatment of eye diseases, retinal function should be monitored carefully.

Activated blood coagulation factor X (FXa) induces angiogenic growth factor expression in human retinal pigment epithelial cells.

PURPOSE: To determine the transcriptional regulation of the blood coagulation factor X (FX) in cultured human retinal pigment epithelial (RPE) cells, and whether the effects of FXa on the chemotaxis and expression of angiogenic growth factors are mediated by autocrine growth factor signaling. METHODS: Alterations in gene expression and secretion of growth factors were determined by real-time RT-PCR and ELISA, respectively. Cellular proliferation and chemotaxis were investigated with a bromodeoxyuridine immunoassay and a Boyden chamber assay, respectively. RESULTS: The gene expression of FX in RPE cells was increased by hypoxia and prostaglandin E(2), and decreased by blood serum, FXa, thrombin, transforming growth factor beta (TGF-ß1), and platelet-derived growth factor (PDGF). The serum-induced downregulation of FX was mediated by thrombin and TGF-ß signaling. FXa induced chemotaxis of RPE cells via activation of the p38 mitogen-activated protein kinase signal transduction pathway. FXa also induced expression of vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF), as well as release of VEGF, bFGF, and TGF-ß1 from RPE cells. The stimulatory effects of FXa on the expression of growth factors and secretion of VEGF were prevented by inhibition of the TGF-ß activin receptor-like kinase, but not by the thrombin inhibitor hirudin. FXa induced phosphorylation of ERK1/2, p38, and Akt proteins. CONCLUSIONS: FXa induces chemotaxis of RPE cells, as well as expression and release of angiogenic growth factors from RPE cells, including VEGF. The effects of FXa on the expression and secretion of VEGF are mediated by autocrine/paracrine TGF-ß signaling.

Transcriptional regulation of aquaporins in the ischemic rat retina: upregulation of aquaporin-9.

PURPOSE/AIM: To determine the transcriptional regulation of retinal aquaporins (AQPs) in rat models of transient and permanent retinal ischemia, and to prove the effects of chemical hypoxia, oxidative stress, glucose, and osmotic alterations on the expression of AQP9 in cultured human retinal pigment epithelium (RPE) cells. MATERIALS AND METHODS: Transient retinal ischemia-reperfusion in rats was induced by elevation of the intraocular pressure for 1 hour. Permanent retinal ischemia was induced by argon laser-induced retinal vein occlusion. The mRNA levels were determined one day after ischemia. RESULTS: Transient and permanent ischemia of the rat retina resulted in downregulation of AQPs 1, 3, 4, 5, 6, 8, and 11 in the RPE and/or neural retina. Pressure-induced transient retinal ischemia-induced upregulation of AQP9 in the neuroretina and RPE, and of AQ12 in the neuroretina. Retinal vein occlusion induced upregulation of AQP0 in the neuroretina and RPE, and of AQP9 and AQP12 in the neuroretina. In cultured human RPE cells, transcriptional expression of AQP9 was stimulated by chemical hypoxia, oxidative stress, VEGF, and high glucose. CONCLUSIONS: The data may suggest that the expression of retinal AQP9 is regulated by metabolic and oxidative stress. Upregulation of AQP9 in RPE cells may prevent lactic acidosis and subretinal edema under ischemic and oxidative stress conditions.

Transcriptional regulation of aquaporin-3 in human retinal pigment epithelial cells.

The expression of aquaporin (AQP) water channels may influence the development of retinal edema. We investigated the transcriptional regulation of AQP3 in cultured human retinal pigment epithelial (RPE) cells. As shown by RT-PCR and immunocytochemistry, cultured RPE cells express AQP3 mRNA and protein. The AQP3 mRNA level in RPE cells was elevated under the following conditions: chemical hypoxia induced by CoCl(2), hyperosmolarity induced by 100 mM NaCl, and upon stimulation of the cultures with PDGF, arachidonic acid, prostaglandin E(2), and blood serum, respectively. Chemical hypoxia increased AQP3 gene expression through MEK/ERK and JNK activation. The hyperosmolarity-, PDGF-, and serum-induced upregulation of AQP3 was prevented by inhibition of the phospholipase A(2), but not by inhibition of the cyclooxygenase. Triamcinolone acetonide prevented the upregulation of AQP3 induced by arachidonic acid and prostaglandin E(2), but not by the other factors tested. It is concluded that AQP3 is transcriptionally activated in RPE cells by various pathogenic factors involved in the development of retinal edema in situ. Activation of phospholipase A(2) is a critical factor which induces AQP3 in RPE cells.