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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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Osmolarity-dependent production of VEGF in RPE cells. The mRNA expression (A, C, E, F) was determined with real-time RT–PCR analysis and is expressed as a fold of isoosmotic unstimulated control. The level of VEGF-A165 protein in the cultured media (B, D) was determined with ELISA and is expressed as a percentage of isoosmotic unstimulated control (100%, corresponding to 169.1±38.9 [B, 6 h], 230.6±55.1 [B, 24 h], and 525.0±90.6 pg/ml VEGF[D], respectively). A. Relative expression level of VEGFA in cells cultured for 2, 6, and 24 h in isoosmotic (- NaCl) and hyperosmotic (+ 100 mM NaCl) media in the absence (- Triam) and presence (+ Triam) of triamcinolone acetonide (50 µM). B. Release of VEGF protein from RPE cells measured under hyperosmotic conditions. The cells were stimulated for 6 and 24 h, respectively, with hyperosmotic medium (+ 100 mM NaCl) in the absence (- Triam) and presence (+ Triam) of triamcinolone acetonide (50 µM). The data are expressed as a percentage of isoosmotic untreated control (100%). C. Effect of hyperosmotic medium (+ 100 mM sucrose) on the expression of VEGFA. D. Effect of hyperosmotic medium (+ 100 mM sucrose) on the release of VEGF protein from RPE cells. E. Dose-dependent effect of high extracellular NaCl on the cellular level of VEGF mRNA. The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. F. Effect of hypoosmotic medium (60% osmolarity) on the expression of VEGFA. Data are means ± SEM of n=4 (C, D, E), 5 (F), 7 (B), and 10 (A) independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05. Significant difference versus NaCl control: ●p<0.05.
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f1: Osmolarity-dependent production of VEGF in RPE cells. The mRNA expression (A, C, E, F) was determined with real-time RT–PCR analysis and is expressed as a fold of isoosmotic unstimulated control. The level of VEGF-A165 protein in the cultured media (B, D) was determined with ELISA and is expressed as a percentage of isoosmotic unstimulated control (100%, corresponding to 169.1±38.9 [B, 6 h], 230.6±55.1 [B, 24 h], and 525.0±90.6 pg/ml VEGF[D], respectively). A. Relative expression level of VEGFA in cells cultured for 2, 6, and 24 h in isoosmotic (- NaCl) and hyperosmotic (+ 100 mM NaCl) media in the absence (- Triam) and presence (+ Triam) of triamcinolone acetonide (50 µM). B. Release of VEGF protein from RPE cells measured under hyperosmotic conditions. The cells were stimulated for 6 and 24 h, respectively, with hyperosmotic medium (+ 100 mM NaCl) in the absence (- Triam) and presence (+ Triam) of triamcinolone acetonide (50 µM). The data are expressed as a percentage of isoosmotic untreated control (100%). C. Effect of hyperosmotic medium (+ 100 mM sucrose) on the expression of VEGFA. D. Effect of hyperosmotic medium (+ 100 mM sucrose) on the release of VEGF protein from RPE cells. E. Dose-dependent effect of high extracellular NaCl on the cellular level of VEGF mRNA. The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. F. Effect of hypoosmotic medium (60% osmolarity) on the expression of VEGFA. Data are means ± SEM of n=4 (C, D, E), 5 (F), 7 (B), and 10 (A) independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05. Significant difference versus NaCl control: ●p<0.05.

Mentions: To determine whether high extracellular NaCl and/or osmolarity alter the expression of VEGF, we stimulated cultured human RPE cells with media that were made hyperosmotic by the addition of 100 mM NaCl or sucrose. As shown in Figure 1A,B, high NaCl induced time-dependent increases in the VEGF mRNA level and secretion of VEGF protein from the cells. Increases in VEGF expression and secretion were also observed after the addition of 100 mM sucrose to the medium (Figure 1C,D). The effect of high extracellular NaCl on the cellular VEGF mRNA level was dose-dependent (Figure 1E). Culturing the cells in a hypoosmotic medium (60% osmolarity) decreased the cellular VEGF mRNA level after 24 h of stimulation (Figure 1F) but did not alter the secretion of VEGF (n=5; not shown). Anti-inflammatory steroids such as triamcinolone acetonide are clinically used for the rapid resolution of retinal edema [40]. Triamcinolone acetonide is known to inhibit the hypoxic secretion of VEGF from RPE cells [41]. Triamcinolone also fully prevented the hyperosmotic increases in VEGF mRNA (Figure 1A) and secretion of VEGF (Figure 1B).


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)

Osmolarity-dependent production of VEGF in RPE cells. The mRNA expression (A, C, E, F) was determined with real-time RT–PCR analysis and is expressed as a fold of isoosmotic unstimulated control. The level of VEGF-A165 protein in the cultured media (B, D) was determined with ELISA and is expressed as a percentage of isoosmotic unstimulated control (100%, corresponding to 169.1±38.9 [B, 6 h], 230.6±55.1 [B, 24 h], and 525.0±90.6 pg/ml VEGF[D], respectively). A. Relative expression level of VEGFA in cells cultured for 2, 6, and 24 h in isoosmotic (- NaCl) and hyperosmotic (+ 100 mM NaCl) media in the absence (- Triam) and presence (+ Triam) of triamcinolone acetonide (50 µM). B. Release of VEGF protein from RPE cells measured under hyperosmotic conditions. The cells were stimulated for 6 and 24 h, respectively, with hyperosmotic medium (+ 100 mM NaCl) in the absence (- Triam) and presence (+ Triam) of triamcinolone acetonide (50 µM). The data are expressed as a percentage of isoosmotic untreated control (100%). C. Effect of hyperosmotic medium (+ 100 mM sucrose) on the expression of VEGFA. D. Effect of hyperosmotic medium (+ 100 mM sucrose) on the release of VEGF protein from RPE cells. E. Dose-dependent effect of high extracellular NaCl on the cellular level of VEGF mRNA. The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. F. Effect of hypoosmotic medium (60% osmolarity) on the expression of VEGFA. Data are means ± SEM of n=4 (C, D, E), 5 (F), 7 (B), and 10 (A) independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05. Significant difference versus NaCl control: ●p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Osmolarity-dependent production of VEGF in RPE cells. The mRNA expression (A, C, E, F) was determined with real-time RT–PCR analysis and is expressed as a fold of isoosmotic unstimulated control. The level of VEGF-A165 protein in the cultured media (B, D) was determined with ELISA and is expressed as a percentage of isoosmotic unstimulated control (100%, corresponding to 169.1±38.9 [B, 6 h], 230.6±55.1 [B, 24 h], and 525.0±90.6 pg/ml VEGF[D], respectively). A. Relative expression level of VEGFA in cells cultured for 2, 6, and 24 h in isoosmotic (- NaCl) and hyperosmotic (+ 100 mM NaCl) media in the absence (- Triam) and presence (+ Triam) of triamcinolone acetonide (50 µM). B. Release of VEGF protein from RPE cells measured under hyperosmotic conditions. The cells were stimulated for 6 and 24 h, respectively, with hyperosmotic medium (+ 100 mM NaCl) in the absence (- Triam) and presence (+ Triam) of triamcinolone acetonide (50 µM). The data are expressed as a percentage of isoosmotic untreated control (100%). C. Effect of hyperosmotic medium (+ 100 mM sucrose) on the expression of VEGFA. D. Effect of hyperosmotic medium (+ 100 mM sucrose) on the release of VEGF protein from RPE cells. E. Dose-dependent effect of high extracellular NaCl on the cellular level of VEGF mRNA. The cells were cultured for 6 h in media that were made hyperosmotic by the addition of 10 to 100 mM NaCl. F. Effect of hypoosmotic medium (60% osmolarity) on the expression of VEGFA. Data are means ± SEM of n=4 (C, D, E), 5 (F), 7 (B), and 10 (A) independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05. Significant difference versus NaCl control: ●p<0.05.
Mentions: To determine whether high extracellular NaCl and/or osmolarity alter the expression of VEGF, we stimulated cultured human RPE cells with media that were made hyperosmotic by the addition of 100 mM NaCl or sucrose. As shown in Figure 1A,B, high NaCl induced time-dependent increases in the VEGF mRNA level and secretion of VEGF protein from the cells. Increases in VEGF expression and secretion were also observed after the addition of 100 mM sucrose to the medium (Figure 1C,D). The effect of high extracellular NaCl on the cellular VEGF mRNA level was dose-dependent (Figure 1E). Culturing the cells in a hypoosmotic medium (60% osmolarity) decreased the cellular VEGF mRNA level after 24 h of stimulation (Figure 1F) but did not alter the secretion of VEGF (n=5; not shown). Anti-inflammatory steroids such as triamcinolone acetonide are clinically used for the rapid resolution of retinal edema [40]. Triamcinolone acetonide is known to inhibit the hypoxic secretion of VEGF from RPE cells [41]. Triamcinolone also fully prevented the hyperosmotic increases in VEGF mRNA (Figure 1A) and secretion of VEGF (Figure 1B).

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