Abstract
Purpose: :
Uncontrolled activation of the alternative complement pathway and secretion of vascular endothelial growth factor (VEGF) are thought to be associated with age-related macular degeneration (AMD). Previously, we have shown that in RPE monolayers, oxidative stress reduced complement inhibition on the cell surface. The resulting increased level of sublytic complement activation resulted in VEGF release, which disrupted the barrier facility of these cells as determined by transepithelial resistance (TER) measurements. Induced rather than basal VEGF release in RPE is thought to be controlled by different mechanisms, including voltage-dependent calcium channel (VDCC) activation and mitogen-activated protein kinases. Here we examined the potential intracellular links between sublytic complement activation and VEGF release in RPE cells challenged with H2O2 and complement-sufficient serum (CSS).
Methods: :
ARPE-19 were grown as monolayers on transwell plates or as confluent cells on coverslips. Effects on RPE tight junctions were determined by transepithelial electrical resistance (TER) measurements. Effects of sublytic MAC on intracellular signaling was analyzed by Western blotting; on VEGF secretion by ELISA; and on membrane potential and ion channel activity by patch-clamp recordings.
Results: :
Treatment with H2O2 + CSS led to a rapid and sustained depolarization of the RPE cells, sufficient to activate VDCC. H2O2 + CSS increased RAS expression and ERK and c-Src phosphorylation, but had no effect on p38 phosphorylation. Either treatment alone had little effect. TER reduction could be attenuated by inhibiting RAS, ERK and c-Src activation, or blocking VDCC, but not by inhibiting p38. All effects on TER were directly correlated with release of VEGF.
Conclusions: :
Sublytic MAC activation results in influx of calcium and depolarizes the cell membrane. Combinatorial analysis of inhibitor effects demonstrated that sublytic complement activation triggers regulated VEGF secretion via two pathways, Src/VDCC and RAS/ERK; but has no effect on constitutive VEGF secretion mediated via p38. Taken together, identifying how sublytic MAC induces VEGF expression and secretion might offer opportunities to selectively inhibit pathological VEGF release only.
Keywords: age-related macular degeneration • retinal pigment epithelium • vascular endothelial growth factor