Activation of L-type Ca2+ Channels is Necessary for Growth Factor-dependent Stimulation of VEGF Secretion by RPE Cells

Abstract

Abstract: : Purpose: Secretion of VEGF (vascular endothelial growth factor) by the RPE is an important mechanism in choroidal neovasularisation (CNV) in age-related macular degeneration. RPE cells express neuroendocrine L-type Ca²⁺ channels known to regulate secretion in many tissues. Purpose of the study is to investigate the regulation of VEGF secretion and the involvement of L-type channels. Methods: VEGF secretion rate was estimated in an in vitro secretion assay. Beginning with the last exchange of the bath solution, the VEGF concentration in bath solution was measured every 4 hours by ELISA. Results: With RPE from eyes without CNV, VEGF concentration rose to constant values after 8-12 hours; VEGF concentrations after: 4 hours: 30.7 ± 12 ng/ml, 8 hours: 66.5 ± 16 ng/ml, 12 hours: 88.8 ± 15.5 ng/ml (all n = 4). L-type Ca²⁺ channel opener (BayK 8644, 10 µM) increased the VEGF secretion rate (after 4 h to 183 ± 35.1 %, n = 3). 0.5 % fetal calf stimulated secretion (after 8h to 170 ± 18 %, n = 4) which was reduced to 99.1 ± 11 % (n = 4) by the L-type channel blocker nifedipine (10 µM). Nifedipine showed no effect on the basic secretion rate. Insulin-like growth factor 1 (after 8 h to 249 ± 1.3 %; n = 2) and bFGF (after 8h to 124 ± 7 %, n = 3) stimulated the secretion. With RPE cells from CNV membranes the VEGF concentration rose after 8h to 247 ± 57 ng/ml (n = 3) . This secretion was stimulated by 0.5 % fetal calf serum (after 8h to 130 ± 14 %; n = 4) and reduced by nifedipine to 110 ± 10 % (n = 4). Conclusions: RPE cells show a basic secretion of VEGF which could be stimulated by growth factors known to be present in CNV membranes. The stimulated VEGF secretion is mediated by activation of L-type Ca²⁺ channels. Since the same mechanisms seem to apply for the VEGF secretion by RPE cells from CNV membranes L-type channels or their regulatory factors represent new targets in the search for a therapy to prevent CNV.

Keywords: choroid: neovascularization • growth factors/growth factor receptors • retinal pigment epithelium