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

Hollborn M, Vogler S, Reichenbach A, Wiedemann P, Bringmann A, Kohen L - Mol. Vis. (2015)

Bottom Line: High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration.The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia.Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.

ABSTRACT

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.

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Regulation of the AQP5 gene expression in RPE cells. The mRNA levels were determined with real-time RT–PCR analysis after stimulation of the cells for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. A. Effect of hyperosmotic culture medium (+ 100 mM NaCl) on the gene expression level of various AQP subtypes (n=8). B. Effect of hyperosmotic medium (+ 100 mM sucrose) on the gene expression of AQP5 (n=4). C. Dose-dependent effect of high extracellular NaCl on the cellular level of AQP5 mRNA (n=4). The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. D. Effect of hypoosmotic medium (60% of control osmolarity) on the gene expression of AQP5 (n=6). E. Effects of high (25 mM) glucose (n=7), CoCl2 (150 µM; n=8), and fetal bovine serum (10%; n=4) on the gene expression of AQP5. F. Regulation of AQP5 expression by triamcinolone acetonide (50 µM) under isoosmotic (control; n=8) and hyperosmotic (+ 100 mM NaCl; n=6) conditions. Data are means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05.
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f5: Regulation of the AQP5 gene expression in RPE cells. The mRNA levels were determined with real-time RT–PCR analysis after stimulation of the cells for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. A. Effect of hyperosmotic culture medium (+ 100 mM NaCl) on the gene expression level of various AQP subtypes (n=8). B. Effect of hyperosmotic medium (+ 100 mM sucrose) on the gene expression of AQP5 (n=4). C. Dose-dependent effect of high extracellular NaCl on the cellular level of AQP5 mRNA (n=4). The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. D. Effect of hypoosmotic medium (60% of control osmolarity) on the gene expression of AQP5 (n=6). E. Effects of high (25 mM) glucose (n=7), CoCl2 (150 µM; n=8), and fetal bovine serum (10%; n=4) on the gene expression of AQP5. F. Regulation of AQP5 expression by triamcinolone acetonide (50 µM) under isoosmotic (control; n=8) and hyperosmotic (+ 100 mM NaCl; n=6) conditions. Data are means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05.

Mentions: The stimulation of cultured RPE cells with a hyperosmotic medium (+ 100 mM NaCl) induced strong time-dependent increases in AQP5 and AQP8 mRNA levels, while the gene expression of further AQPs investigated was not or only moderately altered (Figure 5A). An increase in the AQP5 mRNA level was also observed after the addition of 100 mM sucrose to the medium (Figure 5B). The effect of high extracellular NaCl on the cellular AQP5 mRNA level was dose-dependent (Figure 5C). Extracellular hypoosmolarity decreased the level of AQP5 mRNA (Figure 5D). The AQP5 mRNA level remained largely unaltered in response to oxidative stress induced by the addition of H2O2 (20 µM; n=7; not shown) and in the presence of high (25 mM) glucose (Figure 5E), VEGF (10 ng/ml; n=8), PDGF (10 ng/ml; n=6), IL-1β (10 ng/ml; n=6), TNFα (10 ng/ml; n=5), MMP-2 (10 ng/ml; n=3), arachidonic acid (5 µM; n=4), prostaglandin E2 (10 ng/ml; n=8), thrombin (10 U/ml; n=7), and coagulation factor Xa (1 U/ml; n=6), respectively (data not shown). The AQP5 mRNA level was decreased in the presence of the hypoxia mimetic CoCl2 [44], and strongly reduced in the presence of fetal bovine serum (Figure 5E). The data suggest that the gene expression of AQP5 in RPE cells is relatively specifically regulated by osmotic gradients, hypoxia, and blood serum. Triamcinolone acetonide decreased the AQP5 mRNA level under isoosmotic conditions, but did not prevent the hyperosmotic induction of AQP5 gene expression (Figure 5F).


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

Hollborn M, Vogler S, Reichenbach A, Wiedemann P, Bringmann A, Kohen L - Mol. Vis. (2015)

Regulation of the AQP5 gene expression in RPE cells. The mRNA levels were determined with real-time RT–PCR analysis after stimulation of the cells for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. A. Effect of hyperosmotic culture medium (+ 100 mM NaCl) on the gene expression level of various AQP subtypes (n=8). B. Effect of hyperosmotic medium (+ 100 mM sucrose) on the gene expression of AQP5 (n=4). C. Dose-dependent effect of high extracellular NaCl on the cellular level of AQP5 mRNA (n=4). The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. D. Effect of hypoosmotic medium (60% of control osmolarity) on the gene expression of AQP5 (n=6). E. Effects of high (25 mM) glucose (n=7), CoCl2 (150 µM; n=8), and fetal bovine serum (10%; n=4) on the gene expression of AQP5. F. Regulation of AQP5 expression by triamcinolone acetonide (50 µM) under isoosmotic (control; n=8) and hyperosmotic (+ 100 mM NaCl; n=6) conditions. Data are means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390809&req=5

f5: Regulation of the AQP5 gene expression in RPE cells. The mRNA levels were determined with real-time RT–PCR analysis after stimulation of the cells for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. A. Effect of hyperosmotic culture medium (+ 100 mM NaCl) on the gene expression level of various AQP subtypes (n=8). B. Effect of hyperosmotic medium (+ 100 mM sucrose) on the gene expression of AQP5 (n=4). C. Dose-dependent effect of high extracellular NaCl on the cellular level of AQP5 mRNA (n=4). The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. D. Effect of hypoosmotic medium (60% of control osmolarity) on the gene expression of AQP5 (n=6). E. Effects of high (25 mM) glucose (n=7), CoCl2 (150 µM; n=8), and fetal bovine serum (10%; n=4) on the gene expression of AQP5. F. Regulation of AQP5 expression by triamcinolone acetonide (50 µM) under isoosmotic (control; n=8) and hyperosmotic (+ 100 mM NaCl; n=6) conditions. Data are means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05.
Mentions: The stimulation of cultured RPE cells with a hyperosmotic medium (+ 100 mM NaCl) induced strong time-dependent increases in AQP5 and AQP8 mRNA levels, while the gene expression of further AQPs investigated was not or only moderately altered (Figure 5A). An increase in the AQP5 mRNA level was also observed after the addition of 100 mM sucrose to the medium (Figure 5B). The effect of high extracellular NaCl on the cellular AQP5 mRNA level was dose-dependent (Figure 5C). Extracellular hypoosmolarity decreased the level of AQP5 mRNA (Figure 5D). The AQP5 mRNA level remained largely unaltered in response to oxidative stress induced by the addition of H2O2 (20 µM; n=7; not shown) and in the presence of high (25 mM) glucose (Figure 5E), VEGF (10 ng/ml; n=8), PDGF (10 ng/ml; n=6), IL-1β (10 ng/ml; n=6), TNFα (10 ng/ml; n=5), MMP-2 (10 ng/ml; n=3), arachidonic acid (5 µM; n=4), prostaglandin E2 (10 ng/ml; n=8), thrombin (10 U/ml; n=7), and coagulation factor Xa (1 U/ml; n=6), respectively (data not shown). The AQP5 mRNA level was decreased in the presence of the hypoxia mimetic CoCl2 [44], and strongly reduced in the presence of fetal bovine serum (Figure 5E). The data suggest that the gene expression of AQP5 in RPE cells is relatively specifically regulated by osmotic gradients, hypoxia, and blood serum. Triamcinolone acetonide decreased the AQP5 mRNA level under isoosmotic conditions, but did not prevent the hyperosmotic induction of AQP5 gene expression (Figure 5F).

Bottom Line: High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration.The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia.Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus