¡¡¡¡Angiostatin Downª²regulates VEGF Expression in the Retina of ROP Rats But not in Normal Rats  As overª²expression of VEGF is known as a major cause of vascular hyperª²permeability, we have determined the effect of angiostatin on VEGF expression in ROP rats. Angiostatin (7.5¦Ìg each eye) was injected into the vitreous of the right eyes and PBS into the left eyes of ROP rats at age P14. Twentyª²four hours after the injection, the retina was dissected and pooled for Western blot analysis using an antibody specific for VEGF. Angiostatin injection significantly reduced VEGF levels in the retinas of ROP but did not affect retinal VEGF levels in normal rats (Figure 4), correlating with its effect on vascular permeability.

¡¡¡¡Figure 5  Immunohistochemistry of VEGF in the retina after angiostatin injection  A£ºnegative control ª² retina from the ROP rat with PBS injection in the absence of the antiª²VEGF antibody;B£ºretina from the ROP rat after PBS injection;C£ºretina from ROP rat after angiostatin injection. GCL, ganglion cell layer;  INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. The eye was enucleated at P15 and retinal sections labeled with an antiª²VEGF antibody. The signal was visualized using the ABC method. Note VEGF signal is in brown color

¡¡¡¡Immunohistochemistry using the antiª²VEGF antibody demonª²strated that angiostatin decreased the intensity of VEGF signals in the retina, 24 hours following the injection, when compared to the PBSª²injected eye. The major decrease in VEGF signal occurred in the inner retina after a single dose intravitreal injection of angiostatin (Figure 5).

¡¡¡¡DISCUSSION

¡¡¡¡Angiostatin is a potent angiogenic inhibitor. Its effect has been determined on retinal vascular leakage which is associated with diabetic macular edema, tumor growth and inflamª²mation[4]. Increased vascular permeability in the retina of Streptozotocinª²induced diabetic rats has been reported previously[1]. In this present study, intravitreal injection of angiostatin reduced vascular permeability in the retina of ROP rats in a doseª²dependent manner. Previous studies have shown that angiostatin inhibits retinal neovascularization in the oxygenª²induced retinopathy model[8]. This effect occurred at 1 and 2 days following the angiostatin intravitreal injection. This antiª²angiogenic effect requires higher dose and needs several days to become detectable however the angiostatinª²induced reduction of permeability can be detected as early as 1 day after the injection (Figure 3). Analysis of retinal vasculature showed that angiostatin injection (7.5 ¦Ìg each eye) did not result in any detectable decrease of retinal neovascularization 2 days after the injection when the effect on the reduction of vascular permeability reached a peak. Western blot analysis and immunohistochemistry both showed that angiostatin downª²regulated retinal VEGF expression in the ROP rats but not in ageª²matched normal controls. These results suggest that angiostatinª²induced reduction in vascular permeability is not through its inhibition of neovascularization. This reduction may be ascribed to its downª²regulation of VEGF expression.

¡¡¡¡Oxygenª²induced retinopathy is a widely used model of retinal neovascularization. However, vascular permeability rarely has been studied in this model. The present study showed that oxygenª²induced retinopathy rats also have a transient yet significant increase of vascular permeability in the retina and iris, suggesting the oxygenª²induced retinopathy rat is also a model for vascular permeability studies. PBS injection significantly increased vascular permeability in the retina of normal and oxygenª²induced ROP rats, compared to the eyes without injection. These increases may be due to responses to trauma from the intravitreal injection. Therefore, in the present study, all the control eyes for angiostatin received an injection of the same volume of PBS at the same time as the angiostatin injection, to exclude the interference from the trauma response.

¡¡¡¡Recent studies indicated that one of the proposed mechanisms for the pathogenesis of ROP includes overproduction of the angiogenic growth factors including VEGF[9]. VEGF is also referred to as vascular permeability factor (VPF) based on its potent ability to increase vascular permeability. It has been identified as a major causative factor of retinal vascular hyperª²permeability[10]. Our results are consistent with these previous findings. Another evidence has shown that angiostatin binds to integrins, predominantly ¦Áv¦Â3, on the surface of endothelial cells, but does not induce stress fiber formation, implying that the antiª²angiogenic activity of angiostatin may be through interfering with the ¦Áv¦Â3ª²mediated signaling in endothelial cells[11]. These findings reveal therapeutic potential of angioª²statin in the treatment of retinal neovascularization as well as in the treatment of cancer. This effect is mediated, at least in part, via blockage of VEGF overª²expression under hypoxia.

¡¡¡¡Angiostatin blocks the overª²expression of VEGF in the hypoxic retina as found in ROP but does not decrease the VEGF level in the normal retina. Correlating with this observation, angiostatin only reduces vascular permeability in ROP retina but not in the normal retina. These results suggest that the blockade of VEGF expression in the hypoxic retina is responsible for the angiostatinª²induced reduction of vascular leakage in ROP rats. These studies reveal that angiostatin may have a therapeutic potential in the treatment of retinopathy of prematurity and other diseases with vascular leakage.

¡¡¡¡¡¾²Î¿¼ÎÄÏס¿

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¡¡¡¡8 Meneses PI, Hajjar KA, Berns KI, Duvoisin RM. Recombinant angiostatin prevents retinal neovascularization in a murine proliferative retinopathy model. Gene Ther 2001;8(8):646ª²648

¡¡¡¡9 Wang AY, Chai J, Chen XL. Effect of the dipeptide Argª²Gln on retinopathy of prematurity in mice. Int J Ophthalmol (Guoji Yanke Zazhi) 2008;8(3):442ª²444

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¡¡¡¡11 Tarui T, Miles LA, Takada Y. Specific interaction of angiostatin with integrin alpha(v)beta(3) in endothelial cells. J Biol Chem 2001;276(43): 39562ª²39568

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