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Different effects of angiopoietin-2 in different vascular beds: new vessels are most sensitive

Yuji Oshima^*, Sachiko Oshima^*, Hiroyuki Nambu^*, Shu Kachi^*, Kyoichi Takahashi^*, Naoyasu Umeda^*, JiKui Shen^*, Aling Dong^*, Rajendra S. Apte^*, Elia Duh^*, Sean F. Hackett^*, Godwin Okoye^*, Kazuki Ishibashi^*, James Handa^*, Michele Melia^*, Stanley Wiegand, George Yancopoulos, Donald J. Zack^*, and Peter A. Campochiaro^*,1

^* Departments of Ophthalmology and Neuroscience,
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and
Regeneron Pharmaceuticals, Tarrytown, New York, USA

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

It has been postulated that angiopoietin 2 (Ang2) causes blood vessels to regress when vascular endothelial growth factor (VEGF) levels are low and that in the presence of high levels of VEGF, it stimulates neovascularization (NV). We sought to test this hypothesis in the well-characterized vasculatures of the retina and choroid using double transgenic mice with inducible expression of Ang2.

PRINCIPAL FINDINGS

1. Increased expression of Ang2 has no detectible effect on mature retinal blood vessels and transiently increases the density of developing retinal vessels
The retinal and choroidal vasculatures are well characterized and provide a good system to study the effects of proteins on specific vascular beds. Expression of the protein of interest in transgenic mice with retina-specific promoters provides a way to investigate the effect of increased expression without potential confounding effects from trauma related to injections or effects of viral vectors. Double transgenic mice with doxycycline-inducible expression of the protein of interest provides added control over the timing of expression, allowing investigations at different stages of development and in various model systems; this approach has provided important information regarding activities of VEGF and brain-derived neurotrophic factor in the retina. Tetracycyline response element (TRE)/ang2 mice were generated and crossed with opsin promoter (opsin)/reverse tetracycline transactivator (rtTA), line D mice to give Tet/opsin/ang2 double transgenic mice with doxycycline-inducible expression of Ang2 in photoreceptors. The TRE/ang2 mice were also crossed with CMV/rtTA mice to yield Tet/CMV/ang2 mice with inducible expression of Ang2 in all retinal cells. After 8 weeks of expression of Ang2 in retinal cells, retinas (including retinal vessels) were normal and retinal function assessed by ERG was normal. Therefore, increased expression of Ang2 had no detectable effect on mature retinal vessels.

Retinal blood vessels develop between postnatal day (P) 0 and P18 when the retinal vasculature has assumed the appearance it maintains throughout adulthood. Expression of Ang2 in retinal cells starting soon after birth, caused increased density of retinal capillaries at P11, but despite continued expression of Ang2 there was normalization of the capillary density by P18. This indicates that developing retinal vessels respond to Ang2, but by P18 they have lost that responsiveness.

2. Ang2 expression in ischemic retina stimulates retinal neovascularization, but Ang2 expression after partial resolution of ischemia hastens regression of retinal neovascularization
Exposure of mice to 75% oxygen between P7 and P12 causes regression of retinal vessels resulting in areas of nonperfused retina in which there is up-regulation of VEGF, which promotes retinal NV. Retinal NV peaks between P17 and P21 and then spontaneously regresses due to revascularization of nonperfused retina; the regression of NV is temporally correlated with decreasing levels of vegf mRNA in the retina. In this model of ischemic retinopathy, expression of Ang2 between P12 and P17 when there is substantial retinal ischemia, resulted in a striking increase in retinal neovascularization (Fig. 1 ), whereas delay of Ang2 expression until P20 when there has been amelioration of retinal ischemia, resulted in acceleration of regression of the neovascularization (Fig. 2 ). This indicates that pathologic retinal new vessels, like developing retinal vessels, are sensitive to Ang2, but the effect of Ang2 differs depending on the setting: Ang2 enhances growth of retinal new vessels in the setting of ischemia (and high VEGF), but promotes regression of new vessels when ischemia has resolved (and there is decreased VEGF). This is consistent with the hypothesis that VEGF levels modulate the effects of Ang2, but does not rule out a modulating effect of some other hypoxia-regulated gene product.

View larger version (67K): [in this window] [in a new window]   Figure 1. Increased Ang2 expression during hypoxic period between P12 and P17 causes increased retinal NV in mice with ischemic retinopathy. Double hemizygous Tet/CMV/ang2–5 mice or controls that did not carry both transgenes were placed in 75% oxygen at P7, and then removed to room air at P12 and given daily subcutaneous injections of 0.5 mg/g body weight of doxycycline. At P17, retinal sections stained with Griffonia simplicifolia lectin showed substantially more retinal NV in Tet/CMV/ang2–5 mice (A, C: arrows) compared with controls (B, D: arrows). E) Measurement of the area of NV above the internal limiting membrane showed significantly more NV (*P<0.0001 by unpaired t test) in doxycycline-treated Tet/CMV/ang2–5 mice (ANG2) with ischemic retinopathy (n=10), compared with control mice with ischemic retinopathy (n=14). F) In one litter of mice, the area of NV was measured in one eye, ang2 mRNA was measured in the retina of the fellow eye by RT-PCR, and the results were correlated with the genotype determined by PCR of tail DNA. Double transgenics expressed high levels of ang2 mRNA and had extensive retinal NV, whereas mice that carried only one transgene had low levels of ang2 mRNA and relatively little retinal NV. G)Retinal levels of vegf and ang2 mRNA were elevated in P17 control mice with ischemic retina compared with levels in nonischemic retinas. Doxycycline-treated Tet/CMV/ang2–5 mice with ischemic retinopathy had further elevation of ang2 mRNA, but comparable levels of vegf mRNA compared with ischemic retina from control mice with similar relative differences demonstrated by RT-PCR.

View larger version (98K): [in this window] [in a new window]   Figure 2. Increased Ang2 expression between P20 and P23 causes regression of ischemia-induced retinal NV. Double hemizygous Tet/CMV/ang2–5 mice or controls that did not carry both transgenes were placed in 75% oxygen at P7, and then removed to room air at P12. Six of the mice were killed at P20 and one eye was used to measure the amount of retinal NV and the other was used to measure vegf and ang2 mRNA by RT-PCR. The 20 remaining mice were given daily subcutaneous injections of 0.5 mg/g body weight of doxycycline on P20, P21, and P22, and then killed on P23. The mice were genotyped and one eye was used to measure the amount of retinal NV and the other was used to measure vegf and ang2 mRNA by RT-PCR. Retinal sections stained with Griffonia simplicifolia lectin (GSA) from the mice killed at P20 showed prominent NV on the surface of the retina (A, D: arrows). Eight of the mice killed at P23 were controls, because they lacked one of the transgenes and they showed substantially less NV than the baseline amount at P20 indicating spontaneous regression had occurred (C, F: arrows). Twelve of the mice killed at P23 carried both transgenes and had almost no NV (B, E). Image analysis showed that there was significantly less NV in eyes of P23 double heterozygous transgenics that had been treated with doxycycline compared with eyes of mice that lacked one of the transgenes (G), indicating that high expression of Ang2 in the retina (H) was associated with increased regression of NV.

3. Ang2 inhibits subretinal neovascularization in rho/VEGF transgenic mice and causes regression of choroidal neovascularization at Bruch’s membrane rupture sites
Rupture of Bruch’s membrane in mice results in CNV. Expression of Ang2 in photoreceptors starting right after rupture of Bruch’s membrane suppressed the development of CNV and expression of Ang2 after CNV was established, causing regression of CNV. In this model, there is transient up-regulation of VEGF expression, which may explain the regressive effects of Ang2. However, in transgenic mice with sustained low-level expression of VEGF in photoreceptors, which is sufficient to cause NV to sprout from the deep capillary bed of the retina (where there is constitutive expression of Ang2), coexpression of Ang2 with VEGF resulted in suppression of the NV. This suggests balanced coexpression of Ang2 and VEGF is needed to stimulate neovascularization, and if the balance is disturbed by further increases in Ang2, regression of NV occurs.

CONCLUSIONS AND SIGNIFICANCE

This study demonstrates that modifications are needed in our current thinking regarding the actions of Ang2. Increased expression of Ang2 alone has no detectable effect on mature retinal vessels; despite low constitutive levels of VEGF in the retina, the vessels do not regress. Perhaps mature vessels acquire several different types of survival signals, only some of which are interrupted by Ang2. Coexpression of Ang2 and VEGF is sufficient to cause new vessels to sprout from retinal vessels, suggesting that mature retinal vessels are differentially sensitive to the proangiogenic effects of Ang2 (Fig. 3 ). In contrast, new retinal vessels are sensitive to both the proangiogenic and regressive effects of Ang2, but the latter may predominate, because NV occurring from coexpression of Ang2 and VEGF may be suppressed by additional expression of Ang2. The differential sensitivity of new vessels to the regressive effects of Ang2 suggests that Ang2 may be useful to induce regression of pathologic ocular neovascularization, particularly if combined with VEGF antagonists.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic representation of the interacting effects of Ang2 and VEGF on mature retinal vessels and retinal neovascularization (NV). The top row shows the effects of Ang2 and VEGF on mature retinal vessels. Exposure to Ang2 in the absence of VEGF or in the presence of low levels of VEGF has no effect on retinal vessels. It should be noted that exposure to VEGF in the absence of Ang2 also has no effect. However, exposure to Ang2 combined with moderate or high levels of VEGF results in retinal NV. The bottom row shows the effects of Ang2 and VEGF on retinal NV. Exposure to Ang2 alone or Ang2 and low levels of VEGF results in regression of the NV. Exposure to Ang2 and moderate or high levels of VEGF results in growth of the NV.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2209fje; doi: 10.1096/fj.04-2209fje

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