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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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The hyperosmotic induction of VEGF, but not of AQP5, depends in part on the activity of HIF-1. mRNA levels (A-C, E) were determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. The level of VEGF-A165 protein (D) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in percentages of isoosmotic unstimulated control (100%, corresponding to 383.0±51.1 pg/ml VEGF). A. Effects of chemical hypoxia (150 µM CoCl2) and hyperosmotic medium (+ 100 mM NaCl) on the level of VEGF mRNA (n=6). B. Relative HIF-1α gene expression level in cells treated with hyperosmotic (+ 100 mM NaCl [n=8] and 100 mM sucrose [n=6], respectively) and hypoosmotic (Hypo; 60% osmolarity) media (n=6). C. The hyperosmotic (+ 100 mM NaCl) upregulation of VEGFA was decreased in the presence of an HIF inhibitor (HIF-Inh; 5 µM; n=7). The JAK2 inhibitor AG490 (10 µM; n=5), the STAT3 inhibitor Stattic (1 µM; n=7), and the NF-κB inhibitor caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=7) had no significant effects. D. The hyperosmotic secretion of VEGF was inhibited by the HIF inhibitor (HIF-Inh; 5 µM; n=6), but not by AG490 (10 µM; n=6), Stattic (1 µM; n=6), or caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=6). E. Effects of the HIF inhibitor (HIF-Inh; 5 µM; n=6), AG490 (10 µM; n=6), Stattic (1 µM; n=6), and the NF-κB inhibitor caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=6) on the gene expression of AQP5. Vehicle controls were made with DMSO (1%; n=3 each). Data are means ± SEM obtained in 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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f8: The hyperosmotic induction of VEGF, but not of AQP5, depends in part on the activity of HIF-1. mRNA levels (A-C, E) were determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. The level of VEGF-A165 protein (D) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in percentages of isoosmotic unstimulated control (100%, corresponding to 383.0±51.1 pg/ml VEGF). A. Effects of chemical hypoxia (150 µM CoCl2) and hyperosmotic medium (+ 100 mM NaCl) on the level of VEGF mRNA (n=6). B. Relative HIF-1α gene expression level in cells treated with hyperosmotic (+ 100 mM NaCl [n=8] and 100 mM sucrose [n=6], respectively) and hypoosmotic (Hypo; 60% osmolarity) media (n=6). C. The hyperosmotic (+ 100 mM NaCl) upregulation of VEGFA was decreased in the presence of an HIF inhibitor (HIF-Inh; 5 µM; n=7). The JAK2 inhibitor AG490 (10 µM; n=5), the STAT3 inhibitor Stattic (1 µM; n=7), and the NF-κB inhibitor caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=7) had no significant effects. D. The hyperosmotic secretion of VEGF was inhibited by the HIF inhibitor (HIF-Inh; 5 µM; n=6), but not by AG490 (10 µM; n=6), Stattic (1 µM; n=6), or caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=6). E. Effects of the HIF inhibitor (HIF-Inh; 5 µM; n=6), AG490 (10 µM; n=6), Stattic (1 µM; n=6), and the NF-κB inhibitor caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=6) on the gene expression of AQP5. Vehicle controls were made with DMSO (1%; n=3 each). Data are means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05. Significant difference versus NaCl control: ●p<0.05.

Mentions: The VEGF gene expression induced by chemical hypoxia and hyperosmolarity were not additive (Figure 8A). This suggests the involvement of (at least in part) common mechanisms of the transcriptional activation of VEGF under both conditions. HIF-1 and STAT3 are known transcriptional activators of VEGF [45, 46]. In RPE cells, hyperosmotic challenges increased the gene expression of HIF-1α while hypoosmolarity had no effect (Figure 8B). To reveal whether HIF-1 activity is required for the hyperosmotic induction of AQP5 and VEGF in RPE cells, we tested an HIF inhibitor [47]. The HIF inhibitor decreased the hyperosmotic induction of VEGF by approximately 50% (Figure 8C) and almost completely abrogated the hyperosmotic secretion of VEGF protein (Figure 8D). On the other hand, the Janus kinase (JAK)-2 inhibitor AG490 [48], which inhibits the DNA binding of STAT3; the STAT3 inhibitor Stattic [49]; and the inhibitor of the nuclear transcription factor NF-κB, caffeic acid phenethyl ester [50] had no significant effects on the hyperosmotic induction of VEGF gene transcription (Figure 8C) and the hyperosmotic secretion of VEGF (Figure 8D). The data suggest that the hyperosmotic production of VEGF is in part mediated by HIF-1 but not by STAT3 or NF-κB. In contrast, hyperosmotic AQP5 gene expression was significantly (p<0.05) decreased by the NF-κB inhibitor, while the HIF inhibitor, the JAK2 inhibitor, and the STAT3 inhibitor were without effects (Figure 8E).


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)

The hyperosmotic induction of VEGF, but not of AQP5, depends in part on the activity of HIF-1. mRNA levels (A-C, E) were determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. The level of VEGF-A165 protein (D) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in percentages of isoosmotic unstimulated control (100%, corresponding to 383.0±51.1 pg/ml VEGF). A. Effects of chemical hypoxia (150 µM CoCl2) and hyperosmotic medium (+ 100 mM NaCl) on the level of VEGF mRNA (n=6). B. Relative HIF-1α gene expression level in cells treated with hyperosmotic (+ 100 mM NaCl [n=8] and 100 mM sucrose [n=6], respectively) and hypoosmotic (Hypo; 60% osmolarity) media (n=6). C. The hyperosmotic (+ 100 mM NaCl) upregulation of VEGFA was decreased in the presence of an HIF inhibitor (HIF-Inh; 5 µM; n=7). The JAK2 inhibitor AG490 (10 µM; n=5), the STAT3 inhibitor Stattic (1 µM; n=7), and the NF-κB inhibitor caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=7) had no significant effects. D. The hyperosmotic secretion of VEGF was inhibited by the HIF inhibitor (HIF-Inh; 5 µM; n=6), but not by AG490 (10 µM; n=6), Stattic (1 µM; n=6), or caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=6). E. Effects of the HIF inhibitor (HIF-Inh; 5 µM; n=6), AG490 (10 µM; n=6), Stattic (1 µM; n=6), and the NF-κB inhibitor caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=6) on the gene expression of AQP5. Vehicle controls were made with DMSO (1%; n=3 each). Data are means ± SEM obtained in 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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f8: The hyperosmotic induction of VEGF, but not of AQP5, depends in part on the activity of HIF-1. mRNA levels (A-C, E) were determined with real-time RT–PCR analysis in cells stimulated for 2, 6, and 24 h, and are expressed as folds of isoosmotic unstimulated control. The level of VEGF-A165 protein (D) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in percentages of isoosmotic unstimulated control (100%, corresponding to 383.0±51.1 pg/ml VEGF). A. Effects of chemical hypoxia (150 µM CoCl2) and hyperosmotic medium (+ 100 mM NaCl) on the level of VEGF mRNA (n=6). B. Relative HIF-1α gene expression level in cells treated with hyperosmotic (+ 100 mM NaCl [n=8] and 100 mM sucrose [n=6], respectively) and hypoosmotic (Hypo; 60% osmolarity) media (n=6). C. The hyperosmotic (+ 100 mM NaCl) upregulation of VEGFA was decreased in the presence of an HIF inhibitor (HIF-Inh; 5 µM; n=7). The JAK2 inhibitor AG490 (10 µM; n=5), the STAT3 inhibitor Stattic (1 µM; n=7), and the NF-κB inhibitor caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=7) had no significant effects. D. The hyperosmotic secretion of VEGF was inhibited by the HIF inhibitor (HIF-Inh; 5 µM; n=6), but not by AG490 (10 µM; n=6), Stattic (1 µM; n=6), or caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=6). E. Effects of the HIF inhibitor (HIF-Inh; 5 µM; n=6), AG490 (10 µM; n=6), Stattic (1 µM; n=6), and the NF-κB inhibitor caffeic acid phenethyl ester (CAPE; 1 µg/ml; n=6) on the gene expression of AQP5. Vehicle controls were made with DMSO (1%; n=3 each). Data are means ± SEM obtained in independent experiments performed in triplicate. Significant difference versus isoosmotic unstimulated control: *p<0.05. Significant difference versus NaCl control: ●p<0.05.
Mentions: The VEGF gene expression induced by chemical hypoxia and hyperosmolarity were not additive (Figure 8A). This suggests the involvement of (at least in part) common mechanisms of the transcriptional activation of VEGF under both conditions. HIF-1 and STAT3 are known transcriptional activators of VEGF [45, 46]. In RPE cells, hyperosmotic challenges increased the gene expression of HIF-1α while hypoosmolarity had no effect (Figure 8B). To reveal whether HIF-1 activity is required for the hyperosmotic induction of AQP5 and VEGF in RPE cells, we tested an HIF inhibitor [47]. The HIF inhibitor decreased the hyperosmotic induction of VEGF by approximately 50% (Figure 8C) and almost completely abrogated the hyperosmotic secretion of VEGF protein (Figure 8D). On the other hand, the Janus kinase (JAK)-2 inhibitor AG490 [48], which inhibits the DNA binding of STAT3; the STAT3 inhibitor Stattic [49]; and the inhibitor of the nuclear transcription factor NF-κB, caffeic acid phenethyl ester [50] had no significant effects on the hyperosmotic induction of VEGF gene transcription (Figure 8C) and the hyperosmotic secretion of VEGF (Figure 8D). The data suggest that the hyperosmotic production of VEGF is in part mediated by HIF-1 but not by STAT3 or NF-κB. In contrast, hyperosmotic AQP5 gene expression was significantly (p<0.05) decreased by the NF-κB inhibitor, while the HIF inhibitor, the JAK2 inhibitor, and the STAT3 inhibitor were without effects (Figure 8E).

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