Figure 5
Figure 5. Sema3A produced by hypoxic RGCs prevents retinal EC growth. (A) VEGF and Sema3A release by cultured RGC-5 cells exposed to hypoxia (2%). In the initial 12 hours after hypoxia, VEGF levels rapidly rise ∼ 9-fold, whereas Sema3A remains unaffected. Later, as hypoxic exposure is prolonged, Sema3A levels rise (6-fold at 36 hours and 10-fold at 48 hours). Values represent the fold increase relative to time 0. **P < .01 and ***P < .0001 compared with corresponding time 0. (B) Neuromicrovascular EC proliferation as measured by thymidine incorporation. Incubation of ECs with conditioned medium from RGCs exposed to hypoxia for 12 hours (high VEGF, low Sema3A) caused a 1.7-fold (vehicle) and 2.3-fold (nonspecific shRNA; LV.shGFP) increase in cell number within 24 hours; this effect was abrogated by rSema3A (800 ng/mL). Conversely, ECs treated with conditioned medium from RGCs exposed to hypoxia for 40 hours (low VEGF, high Sema3A), showed a Sema3A-dependent reduction in EC division (5-fold diminution). Knockdown of Sema3A in hypoxia-exposed RGCs using Lv.shSema3A prevented the decrease in EC division compared with vehicle- and Lv.shGFP-treated RGCs. n = 4-6; *P < .05 and **P < .01 compared with vehicle during normoxia (Norm) at time 0. (C) Aortic sprouting more than doubled in explants grown in conditioned medium from vehicle- and LV.shGFP-treated RGCs exposed to 12 hours of hypoxia; this vascular growth was curbed by rSema3A (800 ng/mL). Conditioned medium from RGCs exposed to 40 hours of hypoxia reduced their sprouting by ∼ 60% compared with normoxic medium controls. When Sema3A was knocked down in RGCs, vascular sprouting was doubled compared with Lv.shGFP, underscoring the inhibitory properties of Sema3A toward nascent vessels. Values are represented as the fold change relative to controls. n = 3-6; *P < .05 and **P < .01 compared with vehicle during normoxia (Norm) at time 0. (D) Representative confocal images of the revascularization front (images on left) and high magnification of tip cells (images on right) at OIR P14. The number of filopodia (asterisks) per tip cell was increased 3-fold in Lv.shSema3A animals, whereas contralateral eyes treated with Lv.shGFP showed fewer filopodia and dystrophic tip cells. n = 10; ***P < .0001 compared with value for Lv.shGFP. Scale bars represent 1 mm (C), 50 μm (D left), and 10 μm (D right).

Sema3A produced by hypoxic RGCs prevents retinal EC growth. (A) VEGF and Sema3A release by cultured RGC-5 cells exposed to hypoxia (2%). In the initial 12 hours after hypoxia, VEGF levels rapidly rise ∼ 9-fold, whereas Sema3A remains unaffected. Later, as hypoxic exposure is prolonged, Sema3A levels rise (6-fold at 36 hours and 10-fold at 48 hours). Values represent the fold increase relative to time 0. **P < .01 and ***P < .0001 compared with corresponding time 0. (B) Neuromicrovascular EC proliferation as measured by thymidine incorporation. Incubation of ECs with conditioned medium from RGCs exposed to hypoxia for 12 hours (high VEGF, low Sema3A) caused a 1.7-fold (vehicle) and 2.3-fold (nonspecific shRNA; LV.shGFP) increase in cell number within 24 hours; this effect was abrogated by rSema3A (800 ng/mL). Conversely, ECs treated with conditioned medium from RGCs exposed to hypoxia for 40 hours (low VEGF, high Sema3A), showed a Sema3A-dependent reduction in EC division (5-fold diminution). Knockdown of Sema3A in hypoxia-exposed RGCs using Lv.shSema3A prevented the decrease in EC division compared with vehicle- and Lv.shGFP-treated RGCs. n = 4-6; *P < .05 and **P < .01 compared with vehicle during normoxia (Norm) at time 0. (C) Aortic sprouting more than doubled in explants grown in conditioned medium from vehicle- and LV.shGFP-treated RGCs exposed to 12 hours of hypoxia; this vascular growth was curbed by rSema3A (800 ng/mL). Conditioned medium from RGCs exposed to 40 hours of hypoxia reduced their sprouting by ∼ 60% compared with normoxic medium controls. When Sema3A was knocked down in RGCs, vascular sprouting was doubled compared with Lv.shGFP, underscoring the inhibitory properties of Sema3A toward nascent vessels. Values are represented as the fold change relative to controls. n = 3-6; *P < .05 and **P < .01 compared with vehicle during normoxia (Norm) at time 0. (D) Representative confocal images of the revascularization front (images on left) and high magnification of tip cells (images on right) at OIR P14. The number of filopodia (asterisks) per tip cell was increased 3-fold in Lv.shSema3A animals, whereas contralateral eyes treated with Lv.shGFP showed fewer filopodia and dystrophic tip cells. n = 10; ***P < .0001 compared with value for Lv.shGFP. Scale bars represent 1 mm (C), 50 μm (D left), and 10 μm (D right).

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