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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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Expression of AQP subtypes in freshly isolated and cultured human RPE cells. A. Comparison of the expression levels of AQP1–12 genes in freshly isolated and cultured RPE cells. Bars represent means ± SEM normalized cycle thresholds (ΔCT) required to detect mRNA in real-time RT–PCR. The smaller the ΔCT value, the higher the cellular mRNA level. The values were obtained in four independent preparations of freshly isolated cells from different donors and in three independent cultures using cells from different donors, respectively. Significant difference: *p<0.05. B. Immunolabeling of cultured human RPE cells with antibodies against AQP5 (green) and vimentin (red). Cell nuclei were labeled with Hoechst 33,258 (blue). Right: Negative control cells stained without primary antibodies. Bars, 20 µm.
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f4: Expression of AQP subtypes in freshly isolated and cultured human RPE cells. A. Comparison of the expression levels of AQP1–12 genes in freshly isolated and cultured RPE cells. Bars represent means ± SEM normalized cycle thresholds (ΔCT) required to detect mRNA in real-time RT–PCR. The smaller the ΔCT value, the higher the cellular mRNA level. The values were obtained in four independent preparations of freshly isolated cells from different donors and in three independent cultures using cells from different donors, respectively. Significant difference: *p<0.05. B. Immunolabeling of cultured human RPE cells with antibodies against AQP5 (green) and vimentin (red). Cell nuclei were labeled with Hoechst 33,258 (blue). Right: Negative control cells stained without primary antibodies. Bars, 20 µm.

Mentions: The resolution of osmotic gradients across membranes is facilitated by AQP water channels [30]. We compared the expression levels of AQP genes in RPE cells, which were freshly isolated from human post-mortem donor eyes, and in cultured human RPE cells. In agreement with a previous study [31], we found that both freshly isolated and cultured RPE cells did not contain AQP2 gene transcripts (Figure 4A). Transcripts of AQP4, 6, 7, 10, and 12 genes were detectable in freshly isolated cells, but not in cultured cells (Figure 4A). The expression levels of AQP1, 5, 8, 9, and 11 genes were smaller in cultured cells than in freshly isolated cells, as indicated by the significantly (p<0.05) increased ΔCT values (Figure 4A). The expression levels of AQP0 and AQP3 genes were not different between freshly isolated and cultured cells (Figure 4A). The data indicate that human RPE cells in situ contain gene transcripts of various AQP subtypes, and that the expression of various AQP genes is downregulated in cultured cells compared to freshly isolated cells. It was shown that cultured human RPE cells display immunoreactivities for AQP1, 3, and 9 proteins [31, 43]. We found that the cells also display AQP5 immunoreactivity (Figure 4B).


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)

Expression of AQP subtypes in freshly isolated and cultured human RPE cells. A. Comparison of the expression levels of AQP1–12 genes in freshly isolated and cultured RPE cells. Bars represent means ± SEM normalized cycle thresholds (ΔCT) required to detect mRNA in real-time RT–PCR. The smaller the ΔCT value, the higher the cellular mRNA level. The values were obtained in four independent preparations of freshly isolated cells from different donors and in three independent cultures using cells from different donors, respectively. Significant difference: *p<0.05. B. Immunolabeling of cultured human RPE cells with antibodies against AQP5 (green) and vimentin (red). Cell nuclei were labeled with Hoechst 33,258 (blue). Right: Negative control cells stained without primary antibodies. Bars, 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Expression of AQP subtypes in freshly isolated and cultured human RPE cells. A. Comparison of the expression levels of AQP1–12 genes in freshly isolated and cultured RPE cells. Bars represent means ± SEM normalized cycle thresholds (ΔCT) required to detect mRNA in real-time RT–PCR. The smaller the ΔCT value, the higher the cellular mRNA level. The values were obtained in four independent preparations of freshly isolated cells from different donors and in three independent cultures using cells from different donors, respectively. Significant difference: *p<0.05. B. Immunolabeling of cultured human RPE cells with antibodies against AQP5 (green) and vimentin (red). Cell nuclei were labeled with Hoechst 33,258 (blue). Right: Negative control cells stained without primary antibodies. Bars, 20 µm.
Mentions: The resolution of osmotic gradients across membranes is facilitated by AQP water channels [30]. We compared the expression levels of AQP genes in RPE cells, which were freshly isolated from human post-mortem donor eyes, and in cultured human RPE cells. In agreement with a previous study [31], we found that both freshly isolated and cultured RPE cells did not contain AQP2 gene transcripts (Figure 4A). Transcripts of AQP4, 6, 7, 10, and 12 genes were detectable in freshly isolated cells, but not in cultured cells (Figure 4A). The expression levels of AQP1, 5, 8, 9, and 11 genes were smaller in cultured cells than in freshly isolated cells, as indicated by the significantly (p<0.05) increased ΔCT values (Figure 4A). The expression levels of AQP0 and AQP3 genes were not different between freshly isolated and cultured cells (Figure 4A). The data indicate that human RPE cells in situ contain gene transcripts of various AQP subtypes, and that the expression of various AQP genes is downregulated in cultured cells compared to freshly isolated cells. It was shown that cultured human RPE cells display immunoreactivities for AQP1, 3, and 9 proteins [31, 43]. We found that the cells also display AQP5 immunoreactivity (Figure 4B).

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