A visualization of inhibitory effects on VEGF-mediated signaling pathway in proliferating endothelial cells in...

Vascular endothelial growth factor (VEGF) promotes physiological and pathological retinal angiogenesis by activating the mammalian target of rapamycin complex 1 (mTORC1) pathway in proliferating endothelial cells. This study aimed to visualize the status of the VEGF receptor (VEGFR) pathway in endothelial cells by assessing the phosphorylation of S6 protein (pS6), a downstream marker of mTORC1 activity, in the neonatal mouse retina. Four-day-old mice received subcutaneous injections of rapamycin (mTORC1 inhibitor), PF-4708671 (S6 kinase 1 inhibitor), KRN633 (VEGFR tyrosine kinase inhibitor), or vehicle. The eyes were collected 1, 3, 6, 24, and 48 h after treatment. The vascular density, pS6 distribution, and proliferative activity were evaluated in the retina. pS6 immunoreactivity was detected in developing blood vessels, astrocytes, and microglial cells. Both rapamycin and PF-4708671 almost completely abolished pS6 immunoreactivity in vascular and non-vascular cells 6 h after treatment and thereafter suppressed endothelial cell proliferation before the onset of capillary degeneration. In contrast, KRN633 markedly reduced pS6 immunoreactivity associated with endothelial cells, but not in non-vascular cells at 6 h post-treatment; however, capillary endothelial cell degeneration became apparent at 24 h. These results suggest that VEGFR inhibition disrupts the mTORC1 pathway in endothelial cells within 6 h of treatment, causing endothelial cell degeneration or death in developing retinal blood vessels. Monitoring changes in pS6 immunoreactivity before the onset of endothelial cell degeneration may serve as a valuable method for assessing endothelial responses to VEGF in the retina.