title: Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells.
abstract: BACKGROUND
  Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells.
  
  
  RESULTS
  Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1 signaling and by NFAT5 siRNA, respectively. Hyperosmolarity decreased the viability of the cells; this effect was not altered by exogenous bFGF and HB-EGF. Various vegetable polyphenols (luteolin, quercetin, apigenin) inhibited the hyperosmotic expression of bFGF, HB-EGF, and NFAT5 genes.
  
  
  CONCLUSIONS
  Hyperosmolarity induces transcription of bFGF and HB-EGF genes, and secretion of bFGF from RPE cells. This is in part mediated by autocrine/paracrine TGF-β1 and FGF signaling. It is suggested that high intake of dietary salt resulting in osmotic stress may aggravate neovascular retinal diseases via stimulation of the production of angiogenic factors in RPE cells, independent of hypertension.
authors.affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Veltmann
authors.affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors.forename: Margrit
authors.initials: M
authors.lastname: Hollborn
authors.affiliation: Paul Flechsig Institute of Brain Research, University of Leipzig, Leipzig, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Reichenbach
authors.affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Wiedemann
authors.affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.|Helios Klinikum Aue, Aue, Germany.
authors.forename: Leon
authors.initials: L
authors.lastname: Kohen
authors.affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Bringmann
mesh_terms: D015687:Cell Hypoxia / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D005786:Gene Expression Regulation; D066257:Heparin-binding EGF-like Growth Factor / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008268:Macular Degeneration / Q000503:physiopathology; D018384:Oxidative Stress / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D015398:Signal Transduction / Q000187:drug effects; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D066257:Heparin-binding EGF-like Growth Factor; D010982:Platelet-Derived Growth Factor; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; C075278:platelet-derived growth factor A; D003035:Cobalt; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; C018021:cobaltous chloride