Abstract
Purpose: :
The goal of this study was to extend on our previous work which demonstrated that γ-secretase regulates the inhibitory activity of PEDF in angiogenesis (Cai et al., J Biol Chem 2006) by studying the effect of selective siRNA knockdown of the components of the γ-secretase complex [presenilin1 (PS1), nicastrin (Nct), APH-1 and PEN-2] and determine how this regulates in vitro angiogenesis in choriodal microvascular endothelial cells (CMECs).
Methods: :
siRNA against PS1, Nct, APH-1 or PEN-2 was transfected into bovine choroidal microvascular endothelial cells (CMEC) and the degree of reduction of target proteins was determined by Western blot. Transfected cells and appropriate controls were then exposed to either VEGF-A, PlGF or VEGF-E (100 ng/ml) in the presence of, or absence of, PEDF (100 ng/ml) for up to 24 hours and then seeded into Matrigel. Changes in in vitro tubule length were determined for each condition. Western blots of plasma membrane fractions were probed with antibodies against each component of the γ-secretase complex.
Results: :
siRNA transfection into CMECs silenced γ-secretase components by greater than 80% as confirmed by Western blot analysis. siRNA knockdown of individual γ-secretase components had no effect on in vitro tubule formation by CMECs in either the presence or absence of VEGF-A, PlGF or VEGF-E. In contrast, siRNA knockdown significantly reduced the inhibitory effect of PEDF on VEGF and PlGF-induced tube formation by 62% and 69% respectively within 2 days but had no effect on VEGF-E induced tubule formation, confirming the importance of VEGFR-1 in angiogenesis. siRNA against Nct or PEN-2 demonstrated the greatest reduction in PEDF-induced γ-secretase activity of 43% and 51% respectively compared to PEDF+VEGF alone. Down-regulation of Nct resulted in reduced PS1 processing and activation together with a concomitant loss of APH-1 and PEN-2 from the γ-secretase complex. By contrast, down-regulation of PEN-2 had no significant on the composition of the γ-secretase complex but did reduce its stability.
Conclusions: :
Our results are consistent with the hypothesis that γ-secretase plays a regulatory role in angiogenesis and that specifically targeting γ-secretase components represents an alternative therapeutic strategy for pathological angiogenesis such as choroidal neovascularization.
Keywords: vascular endothelial growth factor • choroid: neovascularization • growth factors/growth factor receptors