Abstract
Purpose::
We have shown that late outgrowth endothelial progenitor cells (OECs) isolated from patients affected or at high risk for developing neovascular AMD have increased proliferation potential compared to control. This study investigated whether VEGFR-2 expression correlated with the observed proliferation potential and whether inhibition of the VEGF/VEGFR-2 signalling pathway could accelerate onset of OEC replicative senescence.
Methods::
Peripheral blood mononuclear cells (PBMNC) were collected from patients with different stages of AMD and control and cultured under defined conditions until outgrowth of OECs was observed. Expression of VEGF receptor-2 (KDR) in expanded OECs was determined by Western blot analysis and correlated to OEC growth characteristics and disease stage. Early passage OECs as well as HUVEC were treated with different VEGF/VEGFR-2 inhibitors and proliferation rate, cell cycle arrest, senescence and telomerase activity were assessed.
Results::
VEGFR-2 expression was related to the number of OEC clusters, the total number of OECs post-expansion and all criteria strongly related to disease category. Small molecule inhibitor SU5416, but not other modalities (other VEGFR-2 inhibitors, neutralizing antibodies, bevacizumab, VEGFR-2 siRNA) inhibited both proliferation and induced irreversible OEC and HUVEC cell cycle arrest and senescence as determined by cell cycle analysis, SA beta-galactosidase staining and p16 and p21 expression analysis. SU5416 but not other inhibitors significantly reduced OEC telomerase activity.
Conclusions::
OEC proliferation potential is increased in patients with or at high risk of developing neovascular AMD and appears to be dependent on VEGF-receptor 2 expression. However, of the VEGF/VEGFR-2 inhibitory treatments analyzed, only SU5416 accelerated OEC senescence and induced irreversible growth arrest. This finding suggests that the observed effect may not exclusively be mediated through the VEGF/VEGFR-2 signalling pathway. Further studies are needed to elucidate the underlying molecular mechanisms and to analyze whether modulation of senescence may be an in vivo target of anti-angiogenic therapy for neovascular AMD.
Keywords: age-related macular degeneration • receptors: pharmacology/physiology • choroid: neovascularization