Expression and regulation of vascular endothelial growth factor in choroidal fibroblasts

Abstract

Subretinal neovascularization is a severe sight-threatening complication into age-related macular degeneration. Previous immunohistochemical studies on surgically removed neovascular membranes have revealed that these membranes, in addition to the neovascular stroma, are comprised of several different cell types such as retinal pigment epithelial (RPE) cells, choroidal fibroblasts and vascular endothelial cells. Since vascular endothelial growth factor (VEGF) potently and specifically induces angiogenesis it was investigated whether VEGF is expressed and/or inducible in choroidal fibroblasts and RPE cells. Choroidal fibroblasts and RPE cells were isolated from human adult post-mortem eyes and expression of VEGF mRNA and protein was measured. By using Northern blotting, both choroidal fibroblasts and RPE cells were found to express VEGF mRNA at low levels. In order to examine whether this VEGF expression was further inducible, the intracellular effector enzyme protein kinase C was activated by phorbol esters. This activation resulted in a prominent increase in VEGF mRNA in choroidal fibroblasts, but not in RPE cells, with a maximal increase detected after 6 h. Elevation of intracellular cyclic AMP levels by forskolin had no clear effect on VEGF mRNA in either cell type. Stimulation with interleukin-1, transforming growth factor beta, tumour necrosis factor alpha and platelet derived growth factor was tested to see if VEGF expression is cytokine inducible. Both interleukin-1 and transforming growth factor beta induced VEGF expression in choroidal fibroblasts although with different time courses. Whereas the transforming growth factor beta effect was transient the interleukin-1 effect was sustained for at least 48 h. None of the cytokines tested affected VEGF expression in RPE cells. By using Western blotting, it was further found that stimulation with interleukin-1 induced VEGF protein expression in choroidal fibroblasts but not in RPE cells. In conclusion, choroidal fibroblasts respond by elevated VEGF mRNA levels after phorbol ester, interleukin-1 and transforming growth factor beta stimulation and elevated VEGF protein levels after phorbol ester and interleukin-1 stimulation suggesting that choroidal fibroblasts may be target cells for increased VEGF synthesis secondary to paracrine cytokine production.