Abstract
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 the maintenance of the choroid and of the retinal pigment epithelium (RPE). VEGF expression is regulated by a plethora of factors which are differently activated by different stimuli. The purpose of this study was to investigate the regulation of constitutive VEGF expression in the RPE.
Methods: :
In order to investigate VEGF regulation, perfusion organ cultures and primary RPE cells of porcine origin were used. VEGF content was evaluated with ELISA and Western Blot. The influence of several molecular factors was assessed by commercially available inhibitors. Toxicity of inhibitors was evaluated in MTT assay.
Results: :
VEGF secretion as measured in RPE/choroid organ culture was diminished after long term (48 h) inhibition of VEGFR-2 by VEGFR-2-antagonist SU1498. It was also diminished after inhibition of phosphatidylinositol 3 kinase (PI3K) by LY294002 for 48 h. Co-application of both substances did not show an additive effect, suggesting that both use the same pathway in an autokrine positive VEGF regulation loop. Inhibition of protein kinase C (PKC) by Bisindolylmaleimide, on the other hand, did not influence VEGF secretion. VEGF expression in primary RPE cell culture displayed a similar pattern, as the inhibition of VEGFR-2 and as well as of PI3K diminished VEGF expression after 48 h. However, in RPE cell culture, an additional effect of PKC inhibition could be found. Inhibition of the transcription factor NFkB diminished VEGF secretion after 6 h (earliest measured time point) and remained diminished during all measured time points (6 h, 24 h, 48 h), suggesting a vital role of NFkB in constitutive VEGF regulation. Inhibition of the transcription factor SP-1 by Mithramycin also displayed effects after 24 h and 48 h. Neither the inhibition of HIF-1 nor Stat3 displayed any influence on constitutive VEGF secretion.
Conclusions: :
Constitutive VEGF in the RPE is regulated by the transcription factor NFkB and may be regulated to lesser extend by the transcription factor SP-1, while Stat3 and HIF-1 do not seem to be involved. Additionally, VEGF secretion is regulated by an autokrine positive loop via VEGFR-2 and PI3K.
Keywords: vascular endothelial growth factor • retinal pigment epithelium