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 5

Reassembled vessels in Bak–/– BaxEC/EC retinas are functional and limit neovascularization and retinal injury.

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Reassembled vessels in Bak–/– BaxEC/EC retinas are functional and limit ...
(A) 48 + 24 RA control and Bak–/– BaxEC/EC retinas perfused i.v. with lectin and stained for PECAM1. (B) Hypoxia visualized by pimonidazole (red) staining (costained with PECAM1, cyan) in 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. (C) Central retina hypoxia in P8 normoxic mice (control, n = 3; Bak–/– BaxEC/EC, n = 3) following 48 hours in high oxygen (control, n = 6; Bak–/– BaxEC/EC, n = 5) or 48 + 24 RA (control, n = 6; Bak–/– BaxEC/EC, n = 4). Two-way ANOVA using Tukey’s multiple-comparisons test. (D) Quantification of VEGFA protein in whole retina extracts from 48 + 24 RA control (n = 4) and Bak–/– BaxEC/EC (n = 4) mice and age-matched normoxic controls (control, n = 4; Bak–/– BaxEC/EC, n = 4). Two-way ANOVA using Tukey’s multiple-comparisons test. (E) Experimental overview of OIR procedure used in F–J. (F–H) Representative examples and quantification of neovascular area in P15 control (n = 8) and Bak–/– BaxEC/EC (n = 5) retinas stained for collagen IV and PECAM1. Yellow lines outline neovascular lesions (F); arrowheads indicate glomerular-like lesions (G). Scale bars: 500 μm (F); 50 μm (G). Student’s 2-tailed t test. (I and J) Representative images and quantification of Müller cell gliosis visualized by GFAP (gray) staining comparing mice subjected to OIR (control, n = 6; Bak–/– BaxEC/EC, n = 6) and age-matched controls raised in room air (normoxia; control, n = 2; Bak–/– BaxEC/EC, n = 2). Isolectin B4 labels ECs (magenta). Scale bars: 100 μm. Two-way ANOVA with Tukey’s multiple-comparisons test. All data are mean ± SEM. Each circle represents 1 animal.