Journal: Molecular Vision

Article Title: Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: Involvement of NFAT5

doi:

Figure Lengend Snippet: 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-A 165 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.

Article Snippet: Recombinant human VEGF-A 165 , platelet-derived growth factor (PDGF)-BB, and tumor necrosis factor (TNF)-α were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Quantitative RT-PCR, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: Involvement of NFAT5

doi:

Figure Lengend Snippet: Involvement of signal transduction pathways in the hyperosmotic induction of VEGF in RPE cells. The mRNA level ( A ) was determined with real-time RT–PCR analysis in cells cultured 6 h under iso- (control) and hyperosmotic conditions (+ 100 mM NaCl), and is expressed as folds of isoosmotic unstimulated control. The level of VEGF-A 165 protein ( B ) was determined with ELISA in the media of cells cultured 24 h under iso- and hyperosmotic conditions, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 281.7±45.8 pg/ml VEGF). The cytosolic protein levels ( C ) were determined by western blot analysis. A. The hyperosmotic upregulation of VEGFA was decreased by the inhibitor of p38 MAPK activation, SB203580 (10 µM; n=6); the inhibitor of ERK1/2 activation, PD98059 (20 µM; n=6); the JNK inhibitor SP600125 (10 µM; n=5); and the inhibitor of PI3K-related kinases, LY294002 (5 µM; n=6), respectively. The inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 µM; n=5), and the inhibitor of the EGF receptor tyrosine kinase, AG1478 (600 nM; n=5), did not inhibit the hyperosmotic upregulation of VEGFA . The vehicle control was made with DMSO (1%; n=3). B. The inhibitors of p38 MAPK (n=5), ERK1/2 (n=5), JNK (n=5), and PI3K (n=5) also decreased the secretion of VEGF induced by hyperosmotic stimulation. C. Stimulation of the cells for 20 min with CoCl 2 (150 µM) or hyperosmotic medium (+ 100 mM NaCl) induced the phosphorylation of p38 and ERK1/2 proteins. PDGF (10 ng/ml) was used as a positive control. Amounts of total proteins are shown above; amounts of phosphorylated proteins are shown below. Similar results were obtained in three independent experiments using cells from different donors. Bars represent 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.

Article Snippet: Recombinant human VEGF-A 165 , platelet-derived growth factor (PDGF)-BB, and tumor necrosis factor (TNF)-α were purchased from R&D Systems (Abingdon, UK).

Techniques: Transduction, Quantitative RT-PCR, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Positive Control

Journal: Molecular Vision

Article Title: Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: Involvement of NFAT5

doi:

Figure Lengend Snippet: 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-A 165 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 CoCl 2 ) 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.

Article Snippet: Recombinant human VEGF-A 165 , platelet-derived growth factor (PDGF)-BB, and tumor necrosis factor (TNF)-α were purchased from R&D Systems (Abingdon, UK).

Techniques: Activity Assay, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing

Journal: Molecular Vision

Article Title: Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: Involvement of NFAT5

doi:

Figure Lengend Snippet: The hyperosmotic induction of AQP5 and VEGF depends on the activity of NFAT5. The mRNA levels ( A, B, D-G ) 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 ( A, B, D ) and NaCl control ( E-G ), respectively. The level of VEGF-A 165 protein ( C, H ) was determined with ELISA in the cultured media of cells stimulated for 24 h, and is expressed in a percentage of isoosmotic unstimulated control (100%, corresponding to 429.5±71.4 pg/ml [ C ] and 324.2±35.1 pg/ml VEGF [ H ], respectively). A-C. The NFAT5 inhibitor rottlerin (2 and 10 µM, respectively) inhibits the hyperosmotic gene expression of AQP5 ( A ; n=7) and VEGF ( B ; n=3), as well as the hyperosmotic secretion of VEGF ( C ; n=5) from RPE cells. Hyperosmolarity was achieved by the addition of 100 mM NaCl to the culture medium. Vehicle control was made with DMSO (1%). D, E. Transfection of RPE cells with NFAT5 siRNA (siNFAT5; 5 nM) results in a reduction of the NFAT5 mRNA level in RPE cells cultured in isoosmotic ( D ; n=5) and hyperosmotic (+ 100 mM NaCl) media ( E ; n=3). Data shown in ( D ) were obtained 48 h after siRNA transfection. Thereafter, the cells were stimulated with a hyperosmotic medium for 2, 6, and 24 h, respectively ( E ). As negative controls, nontargeted siRNA (siNon; 5 nM) and a transfection reagent (TR) without siRNA were used. F, G. Knocking down the gene expression of NFAT5 with siRNA (siNFAT5; 5 nM) reduced the levels of AQP5 ( F ; n=6) and VEGF mRNAs ( G ; n=6) in cells cultured for 24 ( F ) and 6 h ( G ), respectively, in hyperosmotic (+ 100 mM NaCl) medium. As a negative control, nontargeted siRNA (siNon; 5 nM) was used (n=6 each). H. NFAT5 siRNA also reduced the secretion of VEGF induced by hyperosmotic (+ 100 mM NaCl) medium (n=6). 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.

Article Snippet: Recombinant human VEGF-A 165 , platelet-derived growth factor (PDGF)-BB, and tumor necrosis factor (TNF)-α were purchased from R&D Systems (Abingdon, UK).

Techniques: Activity Assay, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Transfection, Negative Control