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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 10, 2006 as doi:10.1096/fj.05-5046fje. |
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* The Departments of Ophthalmology and Neuroscience The Johns Hopkins University School of Medicine Baltimore, Maryland, USA;
Department of Pulmonary Disease and Critical Care, Johns Hopkins University Bayview Medical Center, Baltimore, Maryland, USA; and
Department of Vascular Biology, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan
1Correspondence: Maumenee 719, The Johns Hopkins University School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287-9277, USA. E-mail: pcampo{at}jhmi.edu
SPECIFIC AIMS
Vasohibin is a recently identified protein that is up-regulated in cultured vascular endothelial cells by vascular endothelial growth factor (VEGF) and fibroblast growth factor 2. It inhibits endothelial cell migration, proliferation, and tube formation, and suppresses angiogenesis in chick chorioallantoic membrane, after subcutaneous implantation of matrigel, and in a tumor xenograft model. In this study, we tested the hypothesis that vasohibin functions as a negative feedback inhibitor of VEGF in a well-characterized model of retinal neovascularization.
PRINCIPAL FINDINGS
1. VEGF stimulates the expression of vasohibin in the retina
Oxygen-induced ischemic retinopathy is a useful model for study of hypoxia-induced regulation of VEGF expression. Placement of postnatal day (P) 7 mice into a high oxygen environment results in decreased expression of VEGF and regression of newly developed retinal blood vessels. When mice are returned to room air at P12, the poorly vascularized retina becomes hypoxic and the vegf mRNA level increases, peaks within 12 h, and remains elevated for 
14 days. Measurement of retinal vegf mRNA by quantitative real-time RT-PCR at P13, 24 h after the onset of hypoxia, showed an 4-fold increase compared with the level in P13 control mice. Measurement of vasohibin mRNA in the same samples showed a statistically significant, 2-fold increase in hypoxic retinas compared with control retinas. Compared with intravitreous injection of GFP siRNA at P11, 1 day before mice were moved from 75% oxygen to room air, injection of vegf siRNA resulted in a 3-fold reduction in vegf mRNA in the retinas of P13 mice. It also resulted in nearly a 3-fold reduction in vasohibin mRNA, suggesting that levels of VEGF modulate levels of vasohibin.
Rho/VEGF transgenic mice have increased levels of VEGF in the retina starting at P7. At P16, the level of vegf mRNA was roughly 5-fold higher than that in P16 wild-type mice. Compared with wild-type P16 mice, the level of vasohibin mRNA was 3-fold higher in the retinas of rho/VEGF transgenics. This suggests that increased expression of VEGF in the retina in the absence of hypoxia is sufficient to up-regulate expression of vasohibin.
A polyclonal rabbit antibody that recognizes murine vasohibin was used to localize endogenous vasohibin in mouse retina. At P15, mice that had been reared in room air had normal retinas and no detectable staining for vasohibin (Fig. 1
, middle row, left column), while mice with ischemic retinopathy showed prominent staining that co-localized with CD31, which selectively stains endothelial cells (Fig. 1
, bottom row, right column). This indicates that vasohibin is up-regulated in vascular endothelial cells in ischemic retina.
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2. Vasohibin suppresses VEGF receptor 2 expression in retina, but has no effect on VEGF or VEGF receptor 1
To determine whether vasohibin modulates the expression of VEGF, siRNA targeting vasohibin mRNA was used. Mice were place in 75% oxygen at P7, received an intravitreous injection of vasohibin siRNA in one eye and GFP siRNA in the other eye on P11, and were returned to room air at P12. At P13, the level of vasohibin mRNA in the retina was reduced by almost 3-fold in mice that had been injected with vasohibin siRNA compared with mice injected with GFP siRNA, but the level of vegf mRNA was not significantly different. The effect on mRNA for VEGF receptors was investigated. Retinas with significant knockdown of vasohibin mRNA, showed a significant increase in vegf receptor 2 mRNA, but no significant change in vegf receptor 1 mRNA. This suggests that expression of vasohibin in the retina does not alter the levels of VEGF or VEGF receptor 1, but substantially reduces the level of VEGF receptor 2.
3. Vasohibin suppresses retinal neovascularization in ischemic retina
Vasohibin has been shown to inhibit neovascularization in cornea, subcutaneous matrigel implants, and chick chorioallantoic membrane; however, the angiogenic effects of proteins can vary among vascular beds. To determine the effect of vasohibin on retinal neovascularization, two strategies were used; one was to express human vasohibin in the retinas of mice with oxygen-induced ischemic retinopathy by intraocular injection of an adenoviral vector that expresses vasohibin (AdVasohibin) and the second was to inject recombinant vasohibin. Immunoblots and immunohistochemistry showed that injection of AdVasohibin caused increased levels of vasohibin in the retina. Mice with ischemic retinopathy had intravitreous injection of 109 or 1010 particle units (pu) of AdVasohibin or 109 pu of an empty adenoviral vector (AdNull) at P12 and were killed at P17. Retinas from eyes injected with 109 pu of AdNull showed prominent neovascularization along the surface of the retina (Fig. 2
A, D), while those injected with 109 (Fig. 2B, E
) or 1010 (Fig. 2C, F
) pu of AdVasohibin had little retinal neovascularization. Measurement of the area of retinal neovascularization on the surface of the retina showed that there was significantly less neovascularization in eyes injected with 5 or 10 ng of recombinant human vasohibin compared with eyes injected with PBS (Fig. 2G
) and significantly less neovascularization in eyes injected with 109 or 1010 pu of AdVasohibin compared with eyes injected with 109 AdNull (Fig. 2H
).
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CONCLUSIONS AND SIGNIFICANCE
In this study, we have demonstrated that increased expression of VEGF in the retina, whether it is due to hypoxia or a vegf transgene, is accompanied by increased levels of vasohibin mRNA. In ischemic retina, there is up-regulation of vasohibin in vascular endothelial cells. Knockdown of vegf mRNA by specific siRNAs results in significant reduction of vasohibin mRNA and knockdown of vasohibin mRNA increases ischemia-induced retinal neovascularization. Conversely, increasing levels of vasohibin in the retina by injection of recombinant vasohibin or by gene transfer strongly suppresses ischemia-induced retinal neovascularization. These data support the hypothesis that vasohibin acts as an inhibitor of retinal neovascularization that is up-regulated by VEGF; therefore, vasohibin may act in a negative feedback loop to help suppress neovascularization in the retina (Fig. 3
) as has been postulated for tumors.
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Blunting of the vasohibin rise in ischemic retina by injection of vasohibin siRNA had no significant effect on vegf mRNA levels suggesting that the antiangiogenic effects of vasohibin are not mediated by suppression of VEGF production. However, vasohibin knockdown was accompanied by a significant increase in vegf receptor 2 mRNA, suggesting that the vasohibin negative feedback loop that modulates VEGF activity involves reduction of mRNA for VEGF receptor 2. This could be achieved by inhibiting expression or enhancing degradation of vegf receptor 2 mRNA. There was no effect on vegf receptor 1 mRNA. Interestingly, in cultured endothelial cells, blockade of VEGFR2, but not VEGFR1, suppressed the ability of VEGF to induce vasohibin.
Retinal and choroidal neovascularization are extremely prevalent causes of blindness and are the focus of an intense effort to find selective molecular treatments. This effort has received an important boost from demonstrations that VEGF is an important stimulator for both retinal and choroidal neovascularization. Recently, pegaptanib, an aptamer that binds VEGF, given every 6 wk by intraocular injection, has been demonstrated to slow the rate of vision loss in patients with neovascular AMD. Although the benefits were modest, they confirm that VEGF is an important target. Additional treatments that provide inhibition by different mechanisms are needed. Although the mechanism of the antiangiogenic effect of vasohibin is not completely established, it does not appear to directly antagonize VEGF; therefore, it may have synergistic activity when used in combination with agents that bind VEGF. Additional studies are needed to elucidate the mechanism by which vasohibin works and find ways to exploit its antiangiogenic activity in development of treatments.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5046fje;
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