neovascularization in a murine proliferative retinopathy model. PI Meneses1,2,3, ... retinopathy of prematurity, retinal vein occlusion and, poss- ibly, age-related ...
Gene Therapy (2001) 8, 646–648 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt
BRIEF COMMUNICATION
Recombinant angiostatin prevents retinal neovascularization in a murine proliferative retinopathy model PI Meneses1,2,3, KA Hajjar4, KI Berns1,5 and RM Duvoisin2,6 1
Department of Microbiology, 2Graduate Program in Neuroscience, 4Division of Hematology-Oncology, Departments of Pediatrics and Medicine, and 6Departments of Ophthalmology and Cell Biology, Weill Medical College of Cornell University, New York, NY; and 5Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA
Retinal neovascularization is central to the pathogenesis of proliferative diabetic retinopathy, the leading cause of blindness among the middle-aged population. Angiostatin, a proteolytic fragment of plasminogen is one of the most promising inhibitors of angiogenesis currently in clinical trials. Here we show that recombinant angiostatin can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Because proliferative diabetic retinopathy is a recurrent disease, effective therapy will need to be sustained. Recombinant adeno-associated viruses permit long-term expression of transfected genes; however, they can only accommodate a small insert sequence. Thus, we engineered and tested a shortened recombinant angiostatin derivative containing a
signal sequence to permit secretion. Recombinant protein was purified from the medium of transfected HEK293 cells and injected subcutaneously into treated animals. The retinal vasculature was analyzed in retinal flat mounts and using immunohistochemically stained sections. Both methods demonstrate that this short, secreted form of angiostatin is effective in reducing the development of blood vessels in a nontumor environment and has therapeutic potential for neovascular retinopathies such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion and, possibly, age-related macular degeneration. Gene Therapy (2001) 8, 646–648.
Keywords: angiostatin; neovascularization; proliferative retinopathy
Intraocular neovascularization is a major cause of blindness or partial loss of sight. For example, proliferative diabetic retinopathy and age-related macular degeneration are each the most frequent cause of blindness among the working-age and elderly populations, respectively.1 This proliferation of the microvasculature is thought to result from the pathological expression of angiogenic diffusible factors.2 Retinal neovessels are fragile and rupture easily resulting in vitreal hemorrhage. In addition, they can generate macular traction and cause retinal detachments. Moreover, the permeability of this neovasculature leads to the transudation of serum components and visual loss from macular edema.3 Current therapies to prevent this pathological angiogenesis are directed toward reducing the production of angiogenic factors by ablating parts of the retina using laser photocoagulation or cryotherapy.4 Although these procedures provide some relief, their destructive nature may be counterproductive. Ocular and tumor-induced neovascularization are likely to be similarly growth factor-driven. We reasoned Correspondence: RM Duvoisin, Dyson Vision Research Institute, Box 233, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA 3 Present address: Department of Pathology, Harvard Medical School, Boston, MA 02115, USA Received 30 September 2000; accepted 20 December 2000
that molecules being investigated for their potential to inhibit tumor growth by blocking the formation of new blood vessels could also be effective in preventing retinal neovascularization. Among the most promising inhibitors of angiogenesis currently in clinical trials is angiostatin, a proteolytic fragment of plasminogen.5 Proliferative diabetic retinopathy is a recurrent disease that will require long-term therapy. This may best be achieved by gene transfer using a recombinant adeno-associated virus (AAV). However, since this virus has a small packaging capacity, we engineered and tested a recombinant angiostatin derivative consisting only of the first three kringle domains of human plasminogen (Val79– Ser337) and containing a signal sequence to permit secretion (Figure 1). A carboxyl-terminal hexa-histidine tag was also included to permit purification of the protein from the media of transfected HEK293 cells using a Ni2+ affinity column. A control preparation was obtained from nontransfected HEK293 conditioned media, processed in the same manner as the recombinant K1-3 protein. In a previous study,6 we showed that this recombinant K1-3 angiostatin is capable of inhibiting the growth of human umbilical vein endothelial cells with an IC50 of 50 nm. In immune competent rats, systemic administration of K1-3 angiostatin inhibited the growth of an intracerebral glioma. This growth inhibition was paralleled by a decrease in tumor neovascularization. Here, we used the well-established and reproducible
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Figure 1 Schematic representation of the recombinant K1-3 angiostatin construct. A 789 bp PCR fragment encoding Val79 to Ser337 of human plasminogen was inserted into the pSecTagA vector (Invitrogen, Carlsbad, CA, USA). Fused to angiostatin are an immunoglobulin leader sequence (hatched box), a myc epitope (gray box) and a hexa-histidine sequence (black box). Transcription is initiated at the cytomegalovirus (CMV) promoter and terminated at a bovine growth hormone poly-adenylation site (polyA). The transcription unit can be excised as a 1876 bp NruI–SphI fragment.
murine oxygen-induced retinopathy model7 to examine the potential of angiostatin as a treatment for retinal neovascularization. Seven-day-old C57BL/6 mice were placed in a high oxygen tension chamber (75% O2). After 5 days, the mice were returned to room air, and at P16, P18 or P21, they were deeply anesthetized and their left ventricle was perfused with 2 × 106 molecular weight fluorescein-dextran (Sigma, St Louis, MO, USA) in phosphate buffer saline (PBS). The eyes were enucleated, the retinae were removed, fixed in 10% formalin in PBS for 24 h, mounted flat, and viewed by fluorescence microscopy. As previously observed,7 development of the retinal vasculature is inhibited in mice exposed to high oxygen levels. We found that at P16 the central retina of mice raised in air containing 75% oxygen showed a reduced number of fine branching capillaries around the optic disc compared with mice kept in room air (data not shown). By P18 and P21, neovascular tufts had developed in the hypoperfused area (Figure 2B). Such tufts are present throughout the retina, although they are most prevalent in the mid-periphery, in the region bordering the previously less perfused central retina. For comparison, the retina of mice kept at room air is normal (Figure 2A). To examine the effect of angiostatin on neovascularization, experimental animals were injected subcutaneously twice a day in the back with 4 mg/kg recombinant K1-3 angiostatin starting the day following removal from the hyperoxic chamber (P13). These mice showed a markedly reduced neovascularization and few neovascular tufts. However, the radial vessels often appeared more tortuous than in animals raised in room air (Figure 2C). For a total of 32 animals from three experiments, one eye was studied using dextran-fluorescein perfusion, as described above, while the other was examined histologically. These included nine mice placed in room air with
Figure 3 Retinal vasculature of P21 mice visualized in cross-sections were viewed under light microscopy. (a) Normal retinal vasculature of animals raised in room air. (b) Mice were exposed from P7 to P12 to high oxygen and returned to room air. A large neovessel protruding beyond the ILM is indicated by an arrow. (c) Retinal vasculature of animals exposed to high oxygen and treated with a control prepared from the conditioned media of non-transfected HEK293 cells. Arrows indicate neovascular tufts. (d) Retinal vasculature in mice exposed to high oxygen and treated from P13 to P21 with K1-3 angiostatin. A small vessel is present within the inner plexiform layer (arrow). Scale bar: 20 m. Paraffin-embedded sections were deparaffinized, quenched with hydrogen peroxide, and subjected to antigen retrieval using 750 W microwave pulse (2 min, citrate buffer at pH 6). The sections were then stained with a monoclonal antibody to annexin II (Zymed, South San Francisco, CA, USA), treated with biotinylated secondary antibody and avidin:biotinylated peroxidase according to the manufacturer’s instructions (VectaStain Universal Elite kit, Vector Laboratories, Burlingame, CA, USA), and counterstained with hematoxylin.
no further treatment, seven placed in high oxygen with no further treatment, six placed in oxygen and injected subcutaneously with a control prepared from untransfected HEK293 cells, and 10 placed in oxygen and treated with K1-3 angiostatin. Transverse retinal sections were stained with a monoclonal antibody to annexin II, an endothelial cell marker,8 and counterstained with hematoxylin. Figure 3 shows representative sections from mice that were treated with K1-3 angiostatin, mock-treated or untreated for the duration of the experiments. The internal limiting membrane (ILM) was disrupted in retinae of mice exposed to high oxygen tension. Whereas control animals kept under normal oxygen levels had no cell nuclei on the vitreal side of the ILM (Figure 3a), animals untreated (Figure 3b) or treated with the control material (Figure 3c) showed a number of neovascular tufts (arrows). Fewer such nuclei protruding from the ILM were found in retinae from mice treated daily with
Figure 2 Retinal vasculature of P21 mice visualized by perfusion with fluorescein-dextran and fluorescence microscopy in the flat mount orientation. (A) Normal retinal vasculature of animals raised in room air. (B) Animals exposed from P7 to P12 to high oxygen and returned to room air for 9 days. Microvascular tufts are present in the retina of these animals. (C) Retinal vasculature in mice exposed to high oxygen and treated from P13 to P21 with subcutaneous injections of K1-3 angiostatin. Note the significant reduction in neovascular tufts relative to B. Scale bar: 200 m. Gene Therapy
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as photocoagulation or cryotherapy, are destructive to the retina. We propose that recombinant angiostatin could provide an alternative to, or be used in combination with current surgical treatments. For cancer therapy, the use of angiostatin may be limited by its high dosage requirement. In the case of retinal neovascularization, on the other hand, it may be possible to deliver angiostatin locally using an implanted minipump. Further gene therapy approaches may permit the transfection of retinal vascular cells, which could be induced to secrete recombinant angiostatin into the local vascular milieu. The K1-3 construct tested here is short enough to include a sequence encoding the Rep protein in the recombinant AAV. This could allow site-specific integration of the vector and long-term expression of angiostatin.
Acknowledgements Figure 4 Quantification of retinal neovascularization. (a) Cell nuclei located beyond the ILM were enumerated from an average of five sections per animal as described.7 Animals were treated either with room air (nine mice), high oxygen alone (seven mice), high oxygen plus control preparation (six mice), or high oxygen plus recombinant K1-3 angiostatin (10 mice). The data shown represent mean ± s.e.m. (*P ⬍ 0.001; **P ⬍ 0.05; t-test, two-tailed). (b) Neovascular tufts located beyond the ILM were enumerated as described for panel a. Data shown represent mean ± s.e.m. (* P ⬍ 0.05; t-test, two-tailed).
K1-3 angiostatin (Figure 3d). Occasionally, small vessels were present within the retina, such as the one indicated by an arrow in Figure 3d. These appeared more common than in mice raised at room air and may represent a partial neovascular response. Neovascular tufts and endothelial cell nuclei protruding beyond the ILM into the vitreous were counted as described7 in central retina sections that extended to the ora serrata but did not include the optic nerve. An average of five sections (range 3 to 10 sections) per retina were evaluated. We measured a 64% reduction in neovascular endothelial cell nuclei (Figure 4a), and 50% reduction in neovascular tufts upon recombinant angiostatin treatment (Figure 4b) compared with mocktreated animals. Current treatments for proliferative retinopathy, such
Gene Therapy
We thank Drs Myrna Rosenfeld and Anna Francesconi for discussions and Dr Francis Castellino for providing a plasmid containing human plasminogen cDNA. This research was supported by grants EY09534, EY13101 and AI22251 from the National Institutes of Health.
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