Regulation of constitutive vascular endothelial growth factor secretion in retinal pigment epithelium/choroid organ cultures: p38, nuclear factor kappaB, and the vascular endothelial growth factor receptor-2/phosphatidylinositol 3 kinase pathway

PURPOSE: The retinal pigment epithelium (RPE) is a major source of vascular endothelial growth factor (VEGF) in the eye. Despite the role of VEGF in ocular pathology, VEGF is an important factor in maintaining the choroid and the RPE. Accordingly, the VEGF is constitutively expressed in RPE. In this study, the regulation of constitutive VEGF expression was investigated in an RPE/choroid organ culture. METHODS: To investigate VEGF regulation, RPE/choroid of porcine origin were used. VEGF content was evaluated with enzyme-linked immunosorbent assay. The influence of several molecular factors was assessed with commercially available inhibitors (SU1498, bisindolylmaleimide, LY294002, nuclear factor kappaB [NFkB] activation inhibitor, mithramycin, YC-1, Stattic, SB203580). For toxicity measurements of inhibitors, primary RPE cells of porcine origin were used, and toxicity was evaluated with methyl thiazolyl tetrazolium assay. RESULTS: VEGF secretion as measured in the RPE/choroid organ culture was diminished after long-term (48 h) inhibition of vascular endothelial growth factor receptor-2 by VEGFR-2-antagonist SU1498. VEGF secretion was also diminished after phosphatidylinositol 3 kinase was inhibited by LY294002 for 48 h. Coapplication of the substances did not show an additive effect, suggesting that they use the same pathway in an autocrine-positive VEGF regulation loop. Inhibition of protein kinase C by bisindolylmaleimide, on the other hand, did not influence VEGF secretion in organ culture. Inhibition of the transcription factor SP-1 by mithramycin displayed effects after 24 h and 48 h. Inhibiting hypoxia-inducible factor-1 (HIF-1) and Stat3 did not show any influence on constitutive VEGF secretion. Inhibition of the transcription factor NFkB diminished VEGF secretion after 6 h (earliest measured time point) and remained diminished at all measured time points (24 h, 48 h). The same pattern was found when the inhibitor of mitogen-activated kinase p38 was applied. A combination of NFkB and p38 inhibitors displayed an additive effect, completely abolishing VEGF secretion. CONCLUSIONS: Constitutive VEGF secretion in the RPE/choroid seems to be regulated by the transcription factor NFkB and the mitogen-activated kinase p38 in an independent manner. Constitutive VEGF secretion may be regulated to a lesser extent by the transcription factor SP-1, while Stat3 and hypoxia-inducible factor-1 do not seem to be involved. Additionally, VEGF secretion seems to be regulated long-term by an autocrine positive loop via vascular endothelial growth factor receptor-2 and phosphatidylinositol 3 kinase.