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Comparative Study
. 2013 Feb 1;54(2):918-31.
doi: 10.1167/iovs.12-11291.

Hyperoxia causes regression of vitreous neovascularization by downregulating VEGF/VEGFR2 pathway

Hua Liu  1 , Wenbo Zhang, Zhimin Xu, Robert W Caldwell, Ruth B Caldwell, Steven E Brooks
Affiliations
Comparative Study

Hyperoxia causes regression of vitreous neovascularization by downregulating VEGF/VEGFR2 pathway

Hua Liu et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: Neovascularization (NV) is a sight-threatening complication of retinal ischemia in diabetes, retinal vein occlusion, and retinopathy of prematurity. Current treatment modalities, including laser photocoagulation and repeated intraocular injection of VEGF antagonists, are invasive and not always effective, and may carry side effects. We studied the use of hyperoxia as an alternative therapeutic strategy for regressing established vitreous NV in a mouse model of oxygen-induced ischemic retinopathy.

Methods: Hyperoxia treatment (HT, 75% oxygen) was initiated on postnatal day (P)17 after the onset of vitreous NV. Immunohistochemistry and quantitative PCR were used to assess retinal vascular changes in relation to apoptosis, and expression of VEGFR2 and inflammatory molecules. Effects of intravitreal injections of VEGF-A, VEGF-E, PlGF-1, and VEGF trap were also studied.

Results: HT selectively reduced NV by 70% within 24 hours. It robustly increased the level of cleaved caspase-3 in the vitreous NV between 6 and 18 hours and promoted infiltration of macrophage/microglial cells. The HT-induced apoptosis was preceded by a significant reduction in VEGFR2 expression within the NV and an increase in VEGFR2 within the surrounding neural tissue. Intravitreal VEGF-A and VEGF-E (VEGFR2 agonist) but not PlGF-1 (VEGFR1 agonist) prevented HT-induced apoptosis and regression of NV. In contrast, VEGF trap and VEGFR2 blockers mimicked the effect of HT. However, intravitreal VEGF trap induced increases in inflammatory molecules while HT did not have such unwanted effect.

Conclusions: HT may be clinically useful to specifically treat proliferative NV in ischemic retinopathy.

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Conflict of interest statement

Disclosure: H. Liu, None; W. Zhang, None; Z. Xu, None; R.W. Caldwell, None; R.B. Caldwell, None; S.E. Brooks, None

Figures

Figure 1.
Figure 1.
Hyperoxia treatment induces regression of NV tufts in OIR. OIR mice were treated with hyperoxia (75% oxygen, HT) for 24 hours from P17 to P18 or maintained in room air (OIR). (A) Retinal vessels were stained with isolectin B4 at P18. Upper panel: representative images of retinal flatmounts. Scale bar, 500 μm. Lower panel: high magnification images of NV area taken at 100× by confocal microscopy. Scale bar, 100 μm. (B) NV areas and (C) avascular areas were quantified (n = 8 mice). *P < 0.05 compared with OIR.
Figure 2.
Figure 2.
Regression of NV by hyperoxia is apparent at 12 hours and virtually complete at 24 hours. OIR mice were treated with hyperoxia (75% oxygen, HT) at P17 for 6 to 24 hours. Retinal vessels were stained with isolectin B4 and images were taken at ×100 by confocal microscopy. Representative images show the regression of NV tufts over time (n = 3 mice at each time point). Scale bar, 100 μm.
Figure 3.
Figure 3.
Hyperoxia treatment induces apoptosis in NV tufts and recruitment of macrophage/microglia. OIR mice were treated with hyperoxia (75% oxygen, HT) for 3 to 24 hours at P17. (A) Retinal flatmounts were stained with isolectin B4 (red) and anti-cleaved caspase-3 (green). Representative confocal images of NV tufts are shown (400×, n = 3 mice at each time point). Scale bar, 20 μm. (B) Retinal flatmounts were costained with isolectin B4 (red) for vessels and activated macrophage/microglia, and anti-Iba1 (green) for macrophage/microglia. Representative confocal images of NV tufts are shown (×630, n = 3 mice). Scale bar, 20 μm.
Figure 4.
Figure 4.
VEGF supplement prevents hyperoxia-induced NV apoptosis in OIR mice. (A) OIR mice were treated with hyperoxia (75% oxygen, HT) or room air (control) for 24 hours at P17. VEGF mRNA in the retinas was quantified by qPCR (n = 6 mice). *P < 0.05 compared with control. (B) OIR mice were intravitreally injected with PBS (control) or VEGF (20 ng/eye or 200 ng/eye) at P17 and then treated with hyperoxia (75% oxygen) for 18 hours. Retinal vessels (isolectin B4, red) and apoptotic cells (cleaved caspase-3, green) in flatmounts were shown (×400, n = 3 mice). Scale bar, 20 μm.
Figure 5.
Figure 5.
Blockade of VEGF induces NV apoptosis and regression. OIR mice were intravitreally injected with PBS (control) or VEGF trap (VEGFR1/Fc, 2 μg/eye) at P17. (A) At 18 hours postinjection, retinal flatmounts were stained with isolectin B4 (red) and cleaved caspase-3 (green). Representative confocal images of NV tufts and cleaved caspase-3 are shown (×400, n = 4 mice). Scale bar, 20 μm. (B) At 24 hours postinjection, retinal flatmounts were stained with isolectin B4 (red). Representative images of NV area (×100) are shown. Scale bar, 100 μm. (C) Representative images of retinal flatmounts are shown. NV areas and avascular areas were quantified (n = 6 mice). Scale bar, 500 μm. *P < 0.05 compared with relevant control.
Figure 6.
Figure 6.
Hyperoxia treatment differentially regulates VEGFR2 expression in NV and neural retina. (A) OIR mice were treated with hyperoxia (75% oxygen, HT) for 24 hours at P17. VEGFR2 mRNA in the retinas (left panel) and pooled retinal vessels (right panel) were quantified by qPCR and normalized to OIR mice kept in room air (control; n = 4 to 6 mice). *P < 0.05 compared with control. (B) OIR mice were treated with hyperoxia (75% oxygen, HT) for 6 and 12 hours at P17 and retinal flatmounts were stained with isolectin B4 (red) and anti-VEGFR2 (green). Representative confocal images of NV tufts (marked with white asterisk) in the central retina are shown (×400, n = 4 mice). Scale bar, 20 μm.
Figure 7.
Figure 7.
Hyperoxia treatment does not alter VEGFR2 expression in normal retinas. Normal mice were kept in room air or treated with hyperoxia (75% oxygen, HT) for 12 hours at P17. Retinal flatmounts were stained with isolectin B4 (red) and anti-VEGFR2 (green). Representative confocal images of retinal vessels are shown (×200, n = 3 mice). Scale bar, 50 μm.
Figure 8.
Figure 8.
Activation of VEGFR2 but not VEGFR1 prevents HT-induced NV apoptosis and regression. OIR mice were intravitreally injected with PBS (control); VEGF-A (200 ng/eye); VEGF-E (300 ng/eye); or PlGF-1 (300 ng/eye) at P17, and 1 hour postinjection, mice were treated with hyperoxia (75% oxygen). (A) Mice were treated with hyperoxia for 18 hours and retinal flatmounts were stained with isolectin B4 (red) and cleaved caspase-3 (green). Representative confocal images of NV tufts are shown (×400, n = 4 mice); Scale bar, 20 μm. (B) Mice were treated with hyperoxia for 24 hours and retinal flatmounts were stained with isolectin B4 (red). NV areas and avascular areas were quantified (n = 4 to 6 mice). Scale bar, 500 μm. *P < 0.05 compared with relevant control.
Figure 9.
Figure 9.
Blockade of VEGFR2 is sufficient to induce the apoptosis and regression of NV tufts. (A) OIR mice were intravitreally injected with PBS (control) or VEGFR2 inhibitory peptide (VEGFR2 antagonist, 25 μg/eye) at P17. At 18 hours postinjections, retinas were collected and stained with isolectin B4 (red) and cleaved caspase-3 (green). Representative confocal images of NV tufts are shown (×400, n = 3 mice). Scale bar, 20 μm. (B) At 24 hours postinjections, retinas were collected and stained with isolectin B4 (red). Representative images of retinal flatmounts are shown. Scale bar, 500 μm. NV areas and avascular areas were quantified (n = 4 mice). *P < 0.05 compared with control. (C) OIR mice were intravitreally injected with control siRNA (Con siRNA) or VEGFR2 siRNA (50 pmol/eye) at P17. At 36 hours posttreatment, retinas were collected and stained with isolectin B4 (red). Representative images of retinal flatmounts are shown. Scale bar, 500 μm. NV areas and avascular areas were quantified (n = 6 mice). *P < 0.05 compared with control.
Figure 10.
Figure 10.
VEGF trap, but not hyperoxia treatment induces retinal inflammation. OIR mice were either kept in room air (RA) or treated with hyperoxia therapy (HT), intravitreal PBS (Veh), or VEGF trap (VEGFR1/Fc, 2 μg/eye) at P17. At 24 hours posttreatment, mRNA for inflammatory marker was quantified by qPCR and normalized to OIR mice kept in room air, (n = 8 to 12 mice).
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