Reversible retinal vessel closure from VEGF-induced leukocyte plugging
Yuanyuan Liu, Jikui Shen, Seth D. Fortmann, Jiangxia Wang, Dietmar Vestweber, Peter A. Campochiaro
Yuanyuan Liu, Jikui Shen, Seth D. Fortmann, Jiangxia Wang, Dietmar Vestweber, Peter A. Campochiaro
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Research Article Vascular biology

Reversible retinal vessel closure from VEGF-induced leukocyte plugging

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 3

Turning off VEGF expression or injection of a VEGF-neutralizing protein reduces leukostasis and opens closed vessels.

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Turning off VEGF expression or injection of a VEGF-neutralizing protein ...
On day 3 after intraperitoneal injections of doxycycline on day 0 (50 mg/kg), day 1 (25 mg/kg), and day 2 (25 mg/kg), retinal flat mounts from rhodamine-labeled concanavalin A–perfused (Con A–perfused) Tet/opsin/VEGF mice showed leukocytes adherent to the walls of retinal vessels (A) that appeared to plug the lumen of some vessels (B). Other Tet/opsin/VEGF mice were given 3 injections of doxycycline and then maintained without treatment until day 17 when they were perfused with rhodamine-labeled Con A; retinal flat mounts showed few intravascular leukocytes (C). Tet/opsin/VEGF mice treated in the same manner were perfused with rhodamine-labeled Con A on day 3 or day 17 (n = 6, n=8), and the mean (± SEM) number of intravascular leukocytes per retina was lower on day 17 than on day 3 (D, *P < 0.001 by unpaired t test), indicating that most of the leukocytes had cleared. (E). Fluorescein angiograms obtained on day 3 showed dilation of large retinal vessels between which there was continuous fluorescence interrupted by hypofluorescent regions of nonperfusion (boxes, asterisks). (F). A repeat fluorescein angiogram in the same Tet/opsin/VEGF mouse on day 17 showed that the large retinal vessels were no longer dilated, leakage had resolved allowing better resolution of small vessels, and the previously black areas were filled in with a continuous network of vessels, indicating reopening of previously closed vessels. Magnified images of the large boxed areas in E and F allowed a better visualization of regions of vessel closure induced by high expression of VEGF (G, asterisks), while the same region of retina showed reperfused vessels after levels of VEGF returned to normal (H).Tet/opsin/VEGF mice were given an intravitreous injection of 40 μg aflibercept (n = 5) or PBS (n = 5), and 2 mg/ml doxycycline was added to their drinking water. After 3 days, control mice showed numerous intravascular leukocytes (I), while few leukocytes were seen in vessels of aflibercept-injected eyes (J). The mean (± SEM) number of intravascular leukocytes was significantly lower in eyes injected with aflibercept (K, *P < 0.001 for difference from control by unpaired t test). Fluorescein angiograms in control eyes showed dilation of large retinal vessels, diffuse hyperfluorescence from extravascular leakage, and areas of retinal nonperfusion (L, box), whereas eyes injected with aflibercept showed no vessel dilation, leakage, or retinal nonperfusion (M). Magnification of the boxed area in L clearly shows the dark black patches, indicating closure of retinal vessels in control mice (N, Ctrl, asterisks), while the magnified view of the retinal vessels in M shows the absence of any vessel closure in aflibercept-treated mice (O).