Apelin regulates retinal endothelial cell proliferation through the mTOR pathway. (A) Protein extracts from APLN- and APJ-deficient retinas were analyzed by Western blot for mTOR and ERK-phosphorylation. Total ERK and mTOR were used as loading controls. (B) Quantification of band intensities from Western blots (4 different protein extractions). The ratio of P-mTOR/total mTOR and P-Erk/total Erk in APLN^+/+ and APJ^+/+ was set at 1. (C) Quantification of the branch points in the retinal vasculature following rapamycin injection in wild-type or apln-deficient mice. (D) Quantification of the distance from the optic nerve following rapamycin injection in wild-type or apln-deficient mice. (E-J) PH3 staining (red) of the retina after rapamycin injection shows a decrease in proliferating ECs in wild-type mice, whereas the APLN^−/− ECs are not further affected. Vessels were stained with isolectinB4 (green). The number of PH3 stained NECs is decreased in both wild-type and APLN^−/− mice. ***P < .001. *P < .05. Mann-Whitney test was used. Error bars indicate SEM. Scale bar indicates 80 μm. Images of antibody stainings were acquired on a Leica TCS SP5 confocal microscope using a 20×/0.7 NA objective.