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Reversible retinal vessel closure from VEGF-induced leukocyte plugging
Yuanyuan Liu, … , Dietmar Vestweber, Peter A. Campochiaro
Yuanyuan Liu, … , Dietmar Vestweber, Peter A. Campochiaro
Published September 21, 2017
Citation Information: JCI Insight. 2017;2(18):e95530. https://doi.org/10.1172/jci.insight.95530.
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Research Article Vascular biology

Reversible retinal vessel closure from VEGF-induced leukocyte plugging

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Abstract

Clinical trials in patients with macular edema due to diabetic retinopathy or retinal vein occlusion (RVO) have shown that suppression of VEGF not only improves macular edema, but also reopens closed retinal vessels, prevents progression of vessel closure, and improves retinopathy. In this study, we show the molecular basis for those clinical observations. Increased retinal levels of VEGF in mice cause plugging of retinal vessels with leukocytes, vessel closure, and hypoxia. Suppression of VEGF reduces leukocyte plugging, causing reperfusion of closed vessels. Activation of VEGFR1 contributes to leukocyte recruitment, because it is significantly reduced by an anti-VEGFR1–neutralizing antibody. High VEGF increases transcriptional activity of NF-κB and expression of NF-κB target genes, particularly Vcam1. Injection of an anti-VCAM-1–neutralizing antibody reduces VEGF-induced leukocyte plugging. These data explain the broad range of benefits obtained by VEGF suppression in patients with ischemic retinopathies, provide an important insight into the pathogenesis of RVO and diabetic retinopathy, and suggest that sustained suppression of VEGF early in the course of these diseases may prevent vessel closure, worsening ischemia, and disease progression. This study also identifies VEGFR1 and VCAM-1 as molecular targets whose suppression could supplement VEGF neutralization for treatment of RVO and diabetic retinopathy.

Authors

Yuanyuan Liu, Jikui Shen, Seth D. Fortmann, Jiangxia Wang, Dietmar Vestweber, Peter A. Campochiaro

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Figure 8

VEGF stimulates expression of VCAM-1 in the retina, and neutralization of VCAM-1 reduces VEGF-induced leukostasis.

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VEGF stimulates expression of VCAM-1 in the retina, and neutralization o...
(A) C57BL/6 mice were given an intravitreous injection of PBS or 1 μg VEGF, and qRT-PCR was used to measure retinal mRNA levels for several adhesion molecules. Compared with PBS controls, there was significant elevation of the mean (± SEM) number of mRNA transcripts per 105 Cyclophilin A transcripts for VCAM-1, ICAM-2, integrin β1, and integrin α4 (n in parentheses along x axis, *P < 0.05 by unpaired t tests). (B) In Tet/opsin/VEGF mice, the mean (± SEM) number of mRNA transcripts per 105 Cyclophilin A transcripts for VCAM-1, ICAM-2, integrin β1, and integrin α4 first became significantly elevated after 3 days of doxycycline treatment compared with controls not treated with doxycycline (n = 5 *P < 0.05 by unpaired t tests ). (C–H) One day after C57BL/6 mice were given an intravitreous injection of 1 μg VEGF in one eye and PBS in the fellow eye, ocular frozen sections immunostained for VCAM-1 (red) and Griffonia simplicifolia lectin (green) showed no detectable staining for VCAM-1 in PBS-injected eyes (C–E), but eyes that had been injected with VEGF showed several vessels that stained for VCAM-1 (F–H). (I) Fourteen days after subretinal injection of 0.5 μg NF-κB luciferase reporter vector and 0.25 ng Renilla luciferase plasmid in C57BL/6 mice, an intravitreous injection of 1 μg VEGF was performed. Five days later, the ratio of Firefly/Renilla luciferase was measured in retinal/choroidal homogenates by Dual-Luciferase Reporter Assay. Lines show that the mean relative luciferase activity (n = 6) was significantly greater in VEGF-injected eyes compared with PBS-injected eyes (*P = 0.0138 by unpaired t test). (J) Immunoblots of nuclear fractions from Tet/opsin/VEGF mice treated with doxycycline for 1 (D1), 2 (D2), or 3 days (D3) showed higher levels of NF-κB but equivalent histone 3 in nuclei from retinas with high levels of VEGF compared with those in untreated Tet/opsin/VEGF mice with low levels of VEGF (Ctrl). (K–M) Tet/opsin/VEGF mice (n = 10 for each group) were given an intravenous injection of 100 μg anti-mouse VCAM-1 antibody or rat IgG; after 3 days of doxycycline treatment, there were many adherent leukocytes in retinal vessels of IgG-injected mice (K) and significantly fewer in eyes of anti-VCAM-1–injected mice (L and M) (*P = 0.0013 by unpaired t test). Scale bar: 100 μm.

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