Sema3A produced by hypoxic RGCs repels nascent vessels. (A) Rates of RBMEC migration using a real-time cell analyzer. EC motility was significantly blocked by hypoxia-induced Sema3A (4.2-fold) and rescued by conditioned medium from hypoxic RGC in which Sema3A was silenced (40 hours). n = 4; *P < .05 compared with vehicle at 40 hours of hypoxia. (B) Effect of rSema3A on EC migration rate (over 45 minutes). n = 3; **P < .01 compared with vehicle (Veh). (C) Propensity of Sema3A to deviate (repel) nascent vessels was established using microdeposited Sema3A adjacent to aortic explants from GFP mice. Vascular sprouts invaded vehicle-coated regions but avoided Sema3A-coated zones. (D) RGC-derived Sema3A modulates EC cytoskeletal arrangements and morphology, as demonstrated by time-lapse morphometric analysis of RBMECs subjected to conditioned medium (supplemental Figure 6B). ECs exposed for 45 minutes to conditioned medium from hypoxic RGCs (40 hours) contracted, whereas knockdown of Sema3A in the RGCs largely abrogated this effect. n = 3; **P < .01 compared with vehicle at 40 hours of hypoxia. (E) rSema3A provoked a dose-dependent cellular contraction (22.5%), similar in magnitude to 40 hours of hypoxic conditioned medium. n = 3; **P < .01 compared with vehicle (Veh). (F) Actin stress fibers in ECs. Treatment of ECs with hypoxic conditioned medium from hypoxic retinas resulted in loss of actin stress fibers and collapse of the actin network (as determined by rhodamine-phalloidin staining [red]); knockdown of Sema3A in RGCs abrogated this effect. Therefore, changes in actin are consistent with those on cell shape and movement (panels A-E). Images are representative of 4 experiments. Nuclei are stained with 4′,6-diamidino-2-phenylindole, dihydrochloride (blue). Scale bars represent 20 μm (C) and 50 μm (F).