Different responses of the MIO­M1 Mueller cell line to angiotensin II under hyperglycemic or hypoxic conditions.

Members of the renin-angiotensin aldosterone system (RAAS) are expressed by various retinal tissues including Mueller glial cells. As the RAAS is hypothesized to play an important role in the pathogenesis of diseases that threaten vision, such as diabetic macular edema or retinal vein occlusion, the possible changes induced by exposure of the human cell line MIO-M1, an established model of Mueller cells, to angiotensin II or aldosterone for 6 h under hypoxic and/or hyperglycemic conditions were investigated. The mRNA expression levels of the members of the RAAS were assessed by reverse transcription-quantitative PCR, and the secretion of cytokines was assessed by ELISA. Under hyperglycemic conditions, the mRNA expression levels of the angiotensin-converting enzyme 2 (ACE2), angiotensin II receptors, AT1 and AT2, and the receptor of angiotensin (1-7) MAS1 were significantly higher after exposure to angiotensin II, and the expression of ACE2, AT2, and IL-6 (a marker of inflammation) was significantly increased after treatment with aldosterone; the expression of the other targets investigated remained unchanged. Significantly more IL-6 was secreted by MIO-M1 cells exposed to hyperglycemia and angiotensin. When cells were cultured in a hypoxic environment, additional treatment with aldosterone significantly increased the mRNA expression levels of ACE, but significantly more ACE2 mRNA was expressed in the presence of angiotensin II. Under hypoxic plus hyperglycemic conditions, significantly less ACE but more AT2 was expressed after treatment with angiotensin II, which also led to strongly elevated expression of IL-6. The mRNA expression levels of the angiogenic growth factor VEGF-A and secretion of the encoded protein were notably increased under hypoxic and hypoxic plus hyperglycemic conditions, irrespective of additional treatment with angiotensin II or aldosterone. These findings suggest that angiotensin II induces a pro-inflammatory response in MIO-M1 cells under hyperglycemic conditions despite activation of the counteracting ACE2/MAS1 signaling cascade. However, hypoxia results in an increased expression of angiogenic VEGF-A by these cells, which is not altered by angiotensin II or aldosterone.

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.

Type of culture medium determines properties of cultivated retinal endothelial cells: induction of substantial phenotypic conversion by standard DMEM.

Contradictory behavior of microvascular retinal endothelial cells (REC) - a reliable in vitro model to study retinal diseases - have recently been reported which might result from cultivating the cells in standard DMEM not optimized for this cell type. Therefore, we studied DMEM's effects on phenotype and behavior of immortalized bovine REC. Cells were cultivated in endothelial cell growth medium (ECGM) until a confluent monolayer was reached and then further kept for 1-4 days in ECGM, DMEM, or mixes thereof all supplemented with 5% fetal bovine serum, endothelial cell growth supplement, 90 µg/ml heparin, and 100 nM hydrocortisone. Within hours of cultivation in DMEM, the cell index - measured to assess the cell layer's barrier function - dropped to ~5% of the initial value and only slowly recovered, not only accompanied by stronger expression of HSP70 mRNA and secretion of interleukin-6, but also by lower expressions of tight junction proteins claudin-5, claudin-1 or of the marker of cell type conversion caveolin-1. Altered subcellular localizations of EC-typic claudin-5, vascular endothelial cadherin and von Willebrand factor were also observed. Taken together, all experiments with (retinal) EC cultivated in common DMEM need to be interpreted very cautiously and should at least include phenotypic validation.

Osmotic and hypoxic induction of osteopontin in retinal pigment epithelial cells: Involvement of purinergic receptor signaling.

Purpose: Osteopontin (OPN) is a neuroprotective factor in the retina that improves photoreceptor survival. The aim of the present study was to investigate whether human RPE cells express and respond to OPN. Methods: Hypoxia and chemical hypoxia were induced by cell culture in 0.25% O2 and the addition of CoCl2, respectively. Hyperosmolarity was produced by the addition of 100 mM NaCl or 200 mM sucrose. Gene expression was quantified with real-time reverse transcription (RT)-PCR, and protein secretion was investigated with enzyme-linked immunosorbent assay (ELISA). Nuclear factor of activated T cell 5 (NFAT5) was depleted with siRNA. Results: The acutely isolated RPE cells and the cultured RPE cells expressed OPN. OPN gene expression was induced by hypoxia and hyperosmotic media, as well as by exogenous bFGF. High extracellular NaCl and hypoxia induced secretion of OPN. Hyperosmotic expression of the OPN gene was mediated by the p38 MAPK and ERK1/2 signal transduction pathways, and the transcriptional activities of CREB and NFAT5. The hypoxic expression of the OPN gene was mediated by the PI3K signal transduction pathway and caspase-mediated, necrosis-related pathways. Phospholipases A2 were involved in mediating hyperosmotic and hypoxic OPN gene expression. Autocrine or paracrine P2Y2 receptor signaling induced by extracellular ATP contributed to hyperosmotic expression of the OPN gene whereas activation of A1 receptors by extracellularly formed adenosine contributed to thypoxic OPN gene expression. Autocrine or paracrine VEGF signaling exerted an inhibitory effect on expression of the OPN gene. Exogenous OPN induced expression and secretion of bFGF, but not of VEGF. Conclusions: The data indicated that RPE cells produce and respond to OPN; OPN expression is, in part, induced by the cellular danger signal ATP. RPE-derived neuroprotective factors such as bFGF may contribute to the prosurvival effect of OPN on photoreceptor cells.

Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on KATP channel activation.

Purpose: The expression of aquaporin-8 (AQP8), which plays a crucial role in the maintenance of the cellular fluid and electrolyte balance, was shown to be increased in RPE cells under hyperosmotic conditions. The aim of the present study was to investigate the mechanisms of hyperosmotic AQP8 gene expression and the localization of AQP8 in cultured human RPE cells. Methods: Hyperosmolarity was produced with the addition of 100 mM NaCl or 200 mM sucrose. Hypoxia was induced by cell culture in a 0.2% O2 atmosphere or the addition of the hypoxia mimetic CoCl2. Oxidative stress was induced by the addition of H2O2. Gene expression was determined with real-time RT-PCR analysis. AQP8 protein localization and secretion of VEGF were evaluated with immunocytochemistry, western blotting, and enzyme-linked immunosorbent assay (ELISA). Results: Immunocytochemical and western blot data suggest that the AQP8 protein is mainly located in the mitochondria. Extracellular hyperosmolarity, hypoxia, and oxidative stress induced increases in AQP8 gene expression. Hyperosmotic AQP8 gene expression was reduced by inhibitors of the p38 MAPK and PI3K signal transduction pathways, and by JAK2 and PLA2 inhibitors, and was in part mediated by the transcriptional activity of CREB. Hyperosmotic AQP8 gene expression was also reduced by autocrine/paracrine interleukin-1 signaling, the sulfonylureas glibenclamide and glipizide, which are known inhibitors of KATP channel activation, and a pannexin-blocking peptide. The KATP channel opener pinacidil increased the expression of AQP8 under control conditions. The cells contained Kir6.1 and SUR2B gene transcripts and displayed Kir6.1 immunoreactivity. siRNA-mediated knockdown of AQP8 caused increases in hypoxic VEGF gene expression and secretion and decreased cell viability under control, hyperosmotic, and hypoxic conditions. Conclusions: The data indicate that hyperosmotic expression of AQP8 in RPE cells is dependent on the activation of KATP channels. The data suggest that AQP8 activity decreases the hypoxic VEGF expression and improves the viability of RPE cells which may have impact for ischemic retinal diseases like diabetic retinopathy and age-related macular degeneration.

Osmotic induction of cyclooxygenase-2 in RPE cells: Stimulation of inflammasome activation.

Purpose: Systemic hypertension is a risk factor of age-related macular degeneration, a disease associated with chronic retinal inflammation. The main cause of acute hypertension in the elderly is consumption of dietary salt (NaCl) resulting in increased extracellular osmolarity. The aim of the present study was to determine whether extracellular osmolarity regulates the expression of cyclooxygenase (COX) genes in cultured human retinal pigment epithelial (RPE) cells, and whether COX activity is involved in mediating the osmotic expression of key inflammatory (NLRP3 and IL1B) and angiogenic factor (VEGFA) genes. Methods: Extracellular hyperosmolarity was induced by addition of NaCl or sucrose. Gene expression was determined with real-time reverse transcription (RT)-PCR. Cytosolic interleukin-1ß (IL-1ß) and extracellular vascular endothelial growth factor (VEGF) levels were evaluated with enzyme-linked immunosorbent assay (ELISA). Results: Extracellular hyperosmolarity induced a dose-dependent increase in COX2 gene expression when >10 mM NaCl was added to the culture medium, while COX1 gene expression was increased at higher doses (>50 mM of added NaCl). Extracellular hypo-osmolarity decreased COX2 gene expression. High extracellular osmolarity also induced increases in the COX2 protein level. NaCl-induced expression of COX2 was mediated by various intracellular signal transduction molecules (p38 mitogen-activated protein kinase [p38 MAPK], extracellular signal-regulated kinases 1 and 2 [ERK1/2], and phosphatidylinositol-3 kinase [PI3K]), intracellular calcium signaling involving activation of phospholipase Cγ (PLCγ) and protein kinase Cα/ß (PKCα/ß), and the activity of nuclear factor of activated T cell 5 (NFAT5). Inhibition of fibroblast growth factor (FGF), transforming growth factor-ß (TGF-ß), and interleukin-1 (IL-1) receptor activities decreased NaCl-induced COX2 gene expression. Selective inhibition of COX2 activity decreased osmotic expression of the VEGFA, IL1B, and NLRP3 genes, and blocked the NaCl-induced increase in the cytosolic IL-1ß level. Conclusions: The expression of COX2 in RPE cells is osmoresponsive, and depends on NFAT5. COX2 activity stimulates hyperosmotic expression of angiogenic (VEGFA) and inflammatory factor (IL1B and NLRP3) genes, and activation of the NLRP3 inflammasome in RPE cells.

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.

Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells.

Purpose: Systemic hypertension is a risk factor of neovascular age-related macular degeneration; consumption of dietary salt resulting in extracellular hyperosmolarity is a main cause of hypertension. Extracellular hyperosmolarity was shown to induce expression of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), in RPE cells. The aim of the present study was to determine whether the hyperosmotic expression of growth factor genes in RPE cells is mediated by activator protein-1 (AP-1), and whether c-Fos and c-Jun genes are regulated by extracellular osmolarity. Methods: Hyperosmotic media were made up with the addition of NaCl or sucrose. Gene expression was quantified with real-time reverse transcription (RT)-PCR, and protein secretion was investigated with enzyme-linked immunosorbent assay (ELISA). Nuclear factor of activated T cell 5 (NFAT5) was depleted with siRNA. DNA binding of AP-1 protein was evaluated with electrophoretic mobility shift assay (EMSA). Results: High NaCl and the addition of sucrose triggered expression of the c-Fos gene, but not of the c-Jun gene. High NaCl also increased the levels of c-Fos and phosphorylated c-Jun proteins and the level of DNA binding of AP-1. Hypoosmolarity decreased the expression of the c-Fos and c-Jun genes. NaCl-induced expression of the c-Fos gene was in part mediated by NFAT5. Autocrine/paracrine activation of fibroblast growth factor and adenosine A1 receptors is involved in mediating NaCl-induced expression of the c-Fos gene. Pharmacological inhibition of the AP-1 activity decreased the NaCl-induced expression of the HIF-1α, NFAT5, VEGF, PlGF, and TGF-ß2 genes, and prevented the NaCl-induced secretion of PlGF but not of VEGF. Conclusions: The data indicate that AP-1 is activated in RPE cells in response to extracellular hyperosmolarity and mediates in part via the NaCl-induced expression of VEGF and PlGF, and secretion of PlGF. It is suggested that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in part via activation of AP-1.

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.

Expression and signaling of NGF in the healthy and injured retina.

This review summarizes the present knowledge concerning the retinal localization of the nerve growth factor (NGF), its precursor proNGF, and the receptors TrkA and p75NTR in the developing and mature rodent retina. We further discuss the changes in the expression of NGF and the receptors in experimental models of retinal disorders and diseases like inherited retinitis pigmentosa, retinal detachment, glaucoma, and diabetic retinopathy. Since proNGF is now recognized as a bioactive signaling molecule which induces cell death through p75NTR activation, the role of proNGF in the induction of retinal cell loss under neurodegenerative conditions is also highlighted. In addition, we present the evidences for a potential therapeutic intervention with NGF for the treatment of retinal neurodegenerative diseases. Different strategies have been developed and experimentally tested in mice and rats in order to reduce cell loss and Müller cell gliosis, e.g., increasing the availability of endogenous NGF, administration of exogenous NGF, activation of TrkA, and inhibition of p75NTR. Here, we discuss the several lines of evidence supporting a protective effect of NGF on retinal cell loss, with specific emphasis on photoreceptor and retinal ganglion cell degeneration. A better understanding of the mechanisms underlying the effects of NGF and proNGF in the modulation of neurodegeneration and gliosis in the retina will help to develop efficient therapeutic strategies for various 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.

Impaired Purinergic Regulation of the Glial (Müller) Cell Volume in the Retina of Transgenic Rats Expressing Defective Polycystin-2.

Retinal glial (Müller) cells possess an endogenous purinergic signal transduction cascade which normally prevents cellular swelling in osmotic stress. The cascade can be activated by osmotic or glutamate receptor-dependent ATP release. We determined whether activation of this cascade is altered in Müller cells of transgenic rats that suffer from a slow photoreceptor degeneration due to the expression of a truncated human cilia gene polycystin-2 (CMV-PKD21/703 HA). Age-matched Sprague-Dawley rats served as control. Retinal slices were superfused with a hypoosmotic solution (60 % osmolarity). Müller cells in retinas of PKD21/703 rats swelled immediately in hypoosmotic stress; this was not observed in control retinas. Pharmacological blockade of P2Y1 or adenosine A1 receptors induced osmotic swelling of Müller cells from control rats. The swelling induced by the P2Y1 receptor antagonist was mediated by induction of oxidative-nitrosative stress, mitochondrial dysfunction, production of inflammatory lipid mediators, and a sodium influx from the extracellular space. Exogenous VEGF or glutamate prevented the hypoosmotic swelling of Müller cells from PKD21/703 rats; this effect was mediated by activation of the purinergic signaling cascade. In neuroretinas of PKD21/703 rats, the gene expression levels of P2Y1 and A1 receptors, pannexin-1, connexin 45, NTPDases 1 and 2, and various subtypes of nucleoside transporters are elevated compared to control. The data may suggest that the osmotic swelling of Müller cells from PKD21/703 rats is caused by an abrogation of the osmotic ATP release while the glutamate-induced ATP release is functional. In the normal retina, ATP release and autocrine P2Y1 receptor activation serve to inhibit the induction of oxidative-nitrosative stress, mitochondrial dysfunction, and production of inflammatory lipid mediators, which otherwise will induce a sodium influx and cytotoxic Müller cell swelling under anisoosmotic conditions. Purinergic receptors may represent a target for the protection of retinal glial cells from mitochondrial oxidative stress.

Ischemic regulation of brain-derived neurotrophic factor-mediated cell volume and TrkB expression in glial (Müller) and bipolar cells of the rat retina.

BACKGROUND: Osmotic swelling of neurons and glial cells contributes to retinal edema and neurodegeneration. BDNF, a major neuroprotectant in the retina, was shown to inhibit osmotic swelling of glial (Müller) and bipolar cells in the rat retina; the effect of BDNF on the bipolar cell swelling is mediated by inducing a release of neuroprotective cytokines from Müller cells (Berk et al., Neuroscience 295:175-186, 2015). We determined whether BDNF-mediated cell volume regulation was altered after transient retinal ischemia. METHODS: Retinal slices from the eyes of rats that underwent a 1-h pressure-induced retinal ischemia and from control eyes were superfused with a hypoosmotic solution. RESULTS: Exogenous BDNF prevented osmotic swelling of Müller cells in both control and post-ischemic retinal slices. BDNF also prevented osmotic swelling of bipolar cells in the control retina, but not in the ischemic retina. On the other hand, exogenous bFGF prevented the swelling of both Müller and bipolar cells in the ischemic retina. Freshly isolated Müller cells of control retinas displayed immunoreactivity of truncated but not full-length TrkB. In contrast, Müller cells of post-ischemic retinas displayed immunoreactivity of both TrkB isoforms. Bipolar cells isolated from control and post-ischemic retinas were immunolabeled for both TrkB isoforms. CONCLUSIONS: The data may suggest that the ischemic abrogation of the BDNF effect in bipolar cells is related to altered BDNF receptor expression in Müller cells. Glial upregulation of full-length TrkB may support the survival of Müller cells in the ischemic retina, but may impair the BDNF-induced release of neuroprotective cytokines such as bFGF from Müller cells.

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.