Blocking endothelial apoptosis revascularizes the retina in a model of ischemic retinopathy
Zoe L. Grant, Lachlan Whitehead, Vickie H.Y. Wong, Zheng He, Richard Y. Yan, Abigail R. Miles, Andrew V. Benest, David O. Bates, Claudia Prahst, Katie Bentley, Bang V. Bui, Robert C.A. Symons, Leigh Coultas
Zoe L. Grant, Lachlan Whitehead, Vickie H.Y. Wong, Zheng He, Richard Y. Yan, Abigail R. Miles, Andrew V. Benest, David O. Bates, Claudia Prahst, Katie Bentley, Bang V. Bui, Robert C.A. Symons, Leigh Coultas
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Research Article Angiogenesis

Blocking endothelial apoptosis revascularizes the retina in a model of ischemic retinopathy

Abstract

Aberrant, neovascular retinal blood vessel growth is a vision-threatening complication in ischemic retinal diseases. It is driven by retinal hypoxia frequently caused by capillary nonperfusion and endothelial cell (EC) loss. We investigated the role of EC apoptosis in this process using a mouse model of ischemic retinopathy, in which vessel closure and EC apoptosis cause capillary regression and retinal ischemia followed by neovascularization. Protecting ECs from apoptosis in this model did not prevent capillary closure or retinal ischemia. Nonetheless, it prevented the clearance of ECs from closed capillaries, delaying vessel regression and allowing ECs to persist in clusters throughout the ischemic zone. In response to hypoxia, these preserved ECs underwent a vessel sprouting response and rapidly reassembled into a functional vascular network. This alleviated retinal hypoxia, preventing subsequent pathogenic neovascularization. Vessel reassembly was not limited by VEGFA neutralization, suggesting it was not dependent on the excess VEGFA produced by the ischemic retina. Neutralization of ANG2 did not prevent vessel reassembly, but did impair subsequent angiogenic expansion of the reassembled vessels. Blockade of EC apoptosis may promote ischemic tissue revascularization by preserving ECs within ischemic tissue that retain the capacity to reassemble a functional network and rapidly restore blood supply.

Authors

Zoe L. Grant, Lachlan Whitehead, Vickie H.Y. Wong, Zheng He, Richard Y. Yan, Abigail R. Miles, Andrew V. Benest, David O. Bates, Claudia Prahst, Katie Bentley, Bang V. Bui, Robert C.A. Symons, Leigh Coultas

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Figure 3

Isolated ECs protected from apoptosis rapidly reassemble to revascularize hypoxic retinas.

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Isolated ECs protected from apoptosis rapidly reassemble to revasculariz...
(A and B) PECAM1 staining of control and Bak–/– BaxEC/EC retinas exposed to high oxygen for 48 hours followed by 24 hours in room air (48 + 24 RA). Scale bars: 500 μm (A); 60 μm (B). Arrows indicate sprouting vessels. Boxed areas are enlarged in B (top, yellow box; bottom, pink box). (C) Vessel area in 48 + 24 RA control (n = 11) and Bak–/– BaxEC/EC (n = 12) central retinas. Data for normoxic mice or mice exposed to high oxygen for 48 hours from Figure 1D are included for comparison. Two-way ANOVA using Tukey’s multiple-comparisons test. (D) Central retinal vasculature from control and Bak–/– BaxEC/EC mice exposed to 48 hours of high oxygen or 48 + 24 RA stained for collagen IV (red) and PECAM1 (cyan). Scale bars: 80 μm. (E and F) Network fragmentation (n = 10) and vessel regression (n = 4) in 48 + 24 RA Bak–/– BaxEC/EC central retinas. Data for normoxic mice or mice exposed to 48 hours of high oxygen from Figure 1, F and G, are included for comparison. One-way ANOVA with Tukey’s multiple-comparisons test. Quantitative data from control mice exposed to high oxygen are not shown because there are no central retina capillaries remaining. (G) Static images from live-imaging retinal explants showing vessels reassembling starting 12 hours after return to room air following 48 hours of exposure to high oxygen. Four independent clusters are shown. Time stamp is hh:mm (time 0 = 12 hours after return to room air). Arrows indicate where sprouts form new connections. Scale bars: 50 μm. (H) Sprouting clusters from a 48 + 12 RA Bak–/– BaxEC/EC retina. Scale bar: 50 μm. Arrows indicate filopodial projections. All data are mean ± SEM. Each circle represents 1 animal.