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Intraocular expression of endostatin reduces VEGF-induced retinal vascular permeability, neovascularization, and retinal detachment¹

KYOICHI TAKAHASHI^*, YOSHITSUGU SAISHIN^*, YUMIKO SAISHIN^*, RAQUEL LIMA SILVA^*, YUJI OSHIMA^*, SACHIKO OSHIMA^*, MICHELE MELIA^*, BRIAN PASZKIET, DENNIS ZERBY, MICHAEL J. KADAN, GENE LIAU, MICHAEL KALEKO, SHEILA CONNELLY, TIANCI LUO and PETER A. CAMPOCHIARO^*,2

^* The Departments of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and
Genetic Therapy, A Novartis Company, Gaithersburg, Maryland, USA

²Correspondence: 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

Vascular endothelial growth factor (VEGF) causes several pathologic changes in the retina. Each of these abnormalities, including vascular leakage leading to macular edema, neovascularization, and retinal detachment, are seen in patients with diabetic retinopathy. The major goal of our study was to determine whether intraocular expression of endostatin by gene transfer prevents VEGF-induced pathologies in the retina.

PRINCIPAL FINDINGS

1. Inducible, long-term intraocular expression of endostatin is feasible
For most studies, a bovine immunodeficiency viral vector (BIVendostatin) was used to achieve intraocular expression of endostatin because it provides long-term expression with no identifiable toxicity. In addition, we tested a pair of gutless adenoviral vectors designed to provide tamoxifen-inducible expression of endostatin (InduceAGVendostatin). Mice given a subretinal injection of InduceAGVendostatin showed intense immunostaining for endostatin throughout the retina when given tamoxifen, but not in its absence. Mice given subretinal injection of BIVendostatin showed less intense staining for endostatin, which suggested lower levels than those seen with the inducible AGV system. The less intense staining in BIVendostatin-injected eyes allowed visualization of sites where endostatin was concentrated within the retina; retinal blood vessels were outlined indicating endostatin binding to a component of vessel walls (Fig. 1 ).

View larger version (101K): [in this window] [in a new window]   Figure 1. Expression of endostatin in the retina 4 wk after subretinal injection of BIVendostatin reduces VEGF-stimulated vascular permeability. Adult C57BL/6 mice were given subretinal injections of 1.5 x 106 transducing units (TU) of BIVendostatin in the right eye and 1.5 x 106 TU of BIVnull in the left eye. Four wk after vector injection, mice were killed and ocular frozen sections were immunohistochemically stained for endostatin using HistoMark red, which provides a red reaction product. Eyes injected with BIVendostatin (A, B) showed heavy staining for endostatin in RPE cells (B, large arrows) and throughout the inner nuclear layer with dense staining of the walls of some blood vessels (B, arrows). The linear stained structures in the inner plexiform layer (B, arrowhead) are typical of Muller cell processes. Eyes injected with BIVnull showed reaction product along the internal limiting membrane (C, arrowheads) and Bruch’s membrane (arrows), which is likely to be due to cross-reactivity with collagen XVIII, a known component of these membranes. D) Adult double transgenic IRBP/rtTA-TRE/VEGF mice (n=20) were given a subretinal injection of 1.5 x 106 TU of BIVendostatin in the right eye and 1.5 x 106 TU of BIVnull in the left. Four wk after vector injection, mice were started on 2 mg/mL of doxycycline in their drinking water and on day 3 of treatment retinal vascular permeability was measured using [3H]mannitol as tracer. The retina to lung (RLLR) was significantly reduced in eyes expressing endostatin compared with fellow eyes that had been injected with null vector. Statistical comparisons were made with a paired t test. Bar = 100 µm

2. Endostatin reduces VEGF-induced vascular permeability
Double transgenic mice with doxycycline-inducible expression of VEGF in the retina develop increased retinal vascular permeability after administration of doxycycline. Increased intraocular expression of endostatin using either vector system suppressed VEGF-induced retinal vascular permeability. Several techniques were used to assess retinal vascular leakage, including permeation of [³H]mannitol (Fig. 1D ), fluorescein angiography (Fig. 2A-D ), and assessment of edema by measurement of retinal thickness (Fig. 2E, F ), and each demonstrated an endostatin-induced vascular stabilizing effect. The ratio of [³H]mannitol leakage into the retina vs. leakage into the lung (the retina to lung leakage ratio, RLLR) was significantly less in eyes that expressed both endostatin and VEGF compared with those that expressed only VEGF (Fig. 1D ). Eyes that expressed endostatin and VEGF (Fig. 2 A, C, E) also showed less leakage of intravascular fluorescein from retinal vessels (Fig. 2A, C ) and less thickening of the retina (Fig. 2E ) than eyes that expressed only VEGF (Fig. 2B, D, F ). These findings suggest a new concept: just as angiogenesis may be modulated by a balance between stimulatory and inhibitory factors, the same may be true for vascular permeability.

View larger version (103K): [in this window] [in a new window]   Figure 2. Intraocular BIV-vectored endostatin reduces VEGF-induced fluorescein leakage and retinal thickening. Adult rho/rtTA-TRE/VEGF double transgenic mice were given subretinal injections of 1.5 x 106 transducing units (TU) of BIVendostatin in the right eye and 1.5 x 106 TU of BIVnull in the left. Four wk after vector injection, mice were started on 0.5 mg/mL of doxycycline in their drinking water. Four (A, B) or 7 days (C, D) later, mice were given intraperitoneal injection of 12 µL/g body weight of 1% fluorescein sodium; after 5 min retinas were examined by in vivo fluorescence microscopy and pictures were taken in the left eye, then within 30 s in the right eye. Four days after initiating VEGF expression in the retina with doxycycline, the BIVendostatin-injected eye (A) showed much less fluorescein leakage than the BIVnull-injected eye (B) of the same mouse. In another mouse, 7 days after initiating VEGF expression in the retina with doxycycline, the BIVendostatin-injected eye (C) showed much less fluorescein leakage than the BIVnull-injected eye (D). Seven days after initiating VEGF expression, mice were euthanized and retinal frozen sections were cut through the posterior part of the retina adjacent to the optic nerve in the same location in each eye. The sections were stained with Griffonia simplicifolia lectin, hematoxylin, and eosin. The retina in the BIVnull injected eye (F) is much thicker than the retina in the BIVendostatin-injected eye (E).

3. Endostatin decreases VEGF-induced retinal neovascularization
Increased vascular permeability occurs within hours after the intraocular level of VEGF is increased. If high levels of VEGF are sustained, retinal neovascularization occurs. Double transgenic mice with doxycycline-inducible expression of VEGF develop neovascularization within a few days of starting doxycycline, with time of onset determined by the dose of doxycycline. Intraocular expression of endostatin caused a reduction in VEGF-induced neovascularization. It has previously been demonstrated that high circulating levels of endostatin reduce choroidal neovascularization, a major cause of severe vision loss in patients with age-related macular degeneration. The present study indicates that endostatin also inhibits the development of retinal neovascularization, a major risk factor for severe vision loss in patients with diabetic retinopathy.

4. Endostatin reduces VEGF-induced retinal detachment
The major way that retinal neovascularization leads to severe vision loss in patients with diabetic retinopathy is that it recruits other cells, resulting in fibrovascular scar tissue that contracts and detaches the retina. The same thing happens in double transgenic mice with induced expression of VEGF in the retina; supplementation of drinking water with 2 mg/mL of doxycycline for several days results in very high levels of VEGF in the retina, prominent neovascularization, and retinal detachment. Several agents capable of inhibiting retinal neovascularization in other models are unable to prevent neovascularization and retinal detachment in this model. Intraocular expression of endostatin significantly reduced severe retinal detachment in double transgenics given drinking water containing 2 mg/mL of doxycycline, and therefore endostatin appears to be a strong inhibitor of retinal neovascularization.

CONCLUSIONS

This study provides the first demonstration that endostatin decreases VEGF-induced vascular leakage and raises the possibility that vascular permeability may be modulated by a balance between stimulatory and inhibitory factors, as is postulated to be the case for neovascularization. Endostatin also suppresses VEGF-induced retinal neovascularization and retinal detachment; it therefore provides benefit for each of the three major causes of decreased vision in patients with diabetic retinopathy (Fig. 3 ). Gene transfer can be used to combine long-term intraocular expression of endostatin with inducibility, two features that are quite important for clinical application. This approach should be considered for patients with diabetic retinopathy and other ischemic retinopathies.

View larger version (17K): [in this window] [in a new window]   Figure 3. Schematic representation of potential effects of endostatin and other endogenous inhibitors of neovascularization in diabetic retinopathy.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0824fje; to cite this article, use FASEB J. (March 28, 2003) 10.1096/fj.02-0824fje

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