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`JOURNAL OF CELLULAR PHYSIOLOGY 195:241–248 (2003)
`
`VEGF-TRAPR1R2 Suppresses Choroidal
`Neovascularization and VEGF-Induced Breakdown
`of the Blood–Retinal Barrier
`
`YOSHITSUGU SAISHIN,1 YUMIKO SAISHIN,1 KYOICHI TAKAHASHI,1 RAQUEL LIMA E SILVA,1
`DONNA HYLTON,2 JOHN S. RUDGE,2 STANLEY J. WIEGAND,2
`AND PETER A. CAMPOCHIARO1*
`1The Departments of Ophthalmology and Neuroscience,
`The Johns Hopkins University School of Medicine, Maumenee, Baltimore, Maryland
`2Regeneron Pharmaceuticals, Tarrytown, New York, New York
`
`Vascular endothelial growth factor (VEGF) plays a central role in the development
`of retinal neovascularization and diabetic macular edema. There is also evidence
`suggesting that VEGF is an important stimulator for choroidal neovascularization.
`In this study, we investigated the effect of a specific inhibitor of VEGF, VEGF-
`TRAPR1R2, in models for these disease processes. VEGF-TRAPR1R2 is a fusion
`protein, which combines ligand binding elements taken from the extracellular
`domains of VEGF receptors 1 and 2 fused to the Fc portion of IgG1. Subcutaneous
`injections or a single intravitreous injection of VEGF-TRAPR1R2 strongly suppressed
`choroidal neovascularization in mice with laser-induced rupture of Bruch’s
`membrane. Subcutaneous injection of VEGF-TRAPR1R2 also significantly inhibited
`subretinal neovascularization in transgenic mice that express VEGF in photo-
`receptors. In two models of VEGF-induced breakdown of the blood–retinal barrier
`(BRB), one in which recombinant VEGF is injected into the vitreous cavity and
`one in which VEGF expression is induced in the retina in transgenic mice, VEGF-
`TRAPR1R2 significantly reduced breakdown of the BRB. These data confirm that
`VEGF is a critical stimulus for the development of choroidal neovascularization and
`indicate that VEGF-TRAPR1R2 may provide a new agent for consideration for
`treatment of patients with choroidal neovascularization and diabetic macular
`J. Cell. Physiol. 195: 241–248, 2003. ß 2003 Wiley-Liss, Inc.
`edema.
`
`Ocular neovascularization, consisting of retinal and
`choroidal neovascularization, is an enormous public
`health problem. Retinal neovascularization occurs in
`ischemic retinopathies, the most prevalent of which is
`diabetic retinopathy, the most common cause of severe
`vision loss in young people in developed countries (Klein
`et al., 1984). Choroidal neovascularization complicates
`several diseases in which there are abnormalities of the
`Bruch’s membrane/retinal pigmented epithelial (RPE)
`cell complex, such as age-related macular degeneration
`(AMD), the most common cause of severe vision loss
`in the elderly (The Macular Photocoagulation Study
`Group, 1991). While retinal and choroidal neovascular-
`ization are responsible for the vast majority of severe
`vision loss in Americans, diabetic macular edema is the
`major cause of moderate vision loss (Klein et al., 1984).
`Multiple stimulatory factors may contribute to the
`development of retinal neovascularization, but vascular
`endothelial growth factor (VEGF) plays a critical role.
`Signaling through VEGF receptors is both necessary
`and sufficient for development of retinal neovascular-
`ization (Okamoto et al., 1997; Seo et al., 1999; Ozaki
`et al., 2000). VEGF also causes breakdown of the blood–
`retinal barrier (BRB) (Ozaki et al., 1997), and has been
`implicated in the early breakdown of the BRB that
`occurs in diabetes (Qaum et al., 2001). In addition,
`
`ß 2003 WILEY-LISS, INC.
`
`VEGF is also an important stimulus for choroidal
`neovascularization (Kwak et al., 2000). Therefore,
`antagonizing VEGF is a potentially useful strategy for
`several ocular diseases.
`Many approaches for antagonizing VEGF are being
`considered. One strategy is to inject relatively large
`inhibitors, such as aptamers or FAb fragments of anti-
`VEGF antibodies directly into the eye. Phase I clinical
`
`PAC is the George S. and Dolores Dore Eccles Professor of
`Ophthalmology and Neuroscience.
`
`Contract grant sponsor: Public Health Service; Contract grant
`numbers: EY05951, EY12609, P30EY1765; Contract grant
`sponsor: Foundation Fighting Blindness; Contract grant sponsor:
`Research to Prevent Blindness (Lew R. Wasserman Merit Awards
`and unrestricted grant); Contract grant sponsor: Dr. and Mrs.
`William Lake.
`
`*Correspondence to: Peter A. Campochiaro, Maumenee 719,
`The Johns Hopkins University School of Medicine, 600 N. Wolfe
`Street, Baltimore, MD 21287-9277. E-mail: pcampo@jhmi.edu
`
`Received 30 August 2002; Accepted 20 November 2002
`
`DOI: 10.1002/jcp.10246
`
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`SAISHIN ET AL.
`
`trials testing the safety and tolerability of this approach
`have been completed and phase II and III trials are
`planned or in progress. Preliminary reports suggest
`that inflammation may occur following intraocular
`injection of antibodies or aptamers, but this has not
`been a severe enough problem to discontinue evaluation
`of these approaches (Guyer et al., 2001; Schwartz et al.,
`2001). This approach has some concerns, because
`repeated intraocular injections carry risks of retinal
`detachment and endophthalmitis, and may not be feasi-
`ble depending upon the frequency of injections required.
`Another strategy is to avoid repeated intraocular
`injections by systemic administration of small molecule
`VEGF antagonists (Seo et al., 1999; Kwak et al., 2000;
`Ozaki et al., 2000). There is a theoretical concern that
`some beneficial types of angiogenesis, such as collateral
`formation in ischemic myocardium, may be inhibited.
`But there are no data to support this concern and it is
`equally plausible that systemic inhibition of VEGF could
`have many additional benefits, since angiogenesis has
`been implicated in tumor growth, atherosclerosis, and
`arthritis (for review, see Folkman, 1995). Oral admin-
`istration of VEGF receptor kinase inhibitors results in
`dramatic suppression of retinal and choroidal neovas-
`cularization and is a very promising approach (Seo et al.,
`1999; Kwak et al., 2000; Ozaki et al., 2000). These agents
`are selective, but not specific VEGF antagonists, be-
`cause it is difficult to inhibit VEGF receptor kinases
`without inhibiting homologous kinases such as platelet-
`derived growth factor (PDGF) receptor kinase and c-kit,
`the receptor for stem cell factor (Fabbro et al., 1999; Bold
`et al., 2000; Drevs et al., 2000; Wood et al., 2000). The
`effects of these additional activities are unknown and
`while they are being investigated, it is prudent to con-
`sider and test more selective VEGF inhibitors.
`Soluble VEGF receptors provide a very specific way to
`antagonize VEGF, and several studies have demon-
`strated that the extracellular domain of VEGF receptor
`1 (VEGF-R1) has antiangiogenic activity (Goldman
`et al., 1998; Kong et al., 1998; Honda et al., 2000; Shiose
`et al., 2000; Takayama et al., 2000; Lai et al., 2001;
`Mahasreshti et al., 2001; Bainbridge et al., 2002; Lai
`et al., 2002). A disadvantage of soluble VEGF-R1 is that
`it is cleared fairly rapidly. Pharmacokinetic properties
`can be improved by linking the ligand binding domains
`of VEGF receptors to the Fc portion of IgG, which slows
`clearance by conferring the long circulating half-life of
`an antibody to the chimeric molecule. A potential trade
`off is that the relatively large size of such constructs
`could limit tissue penetration from the systemic circula-
`tion, which is a particularly important consideration
`for treatment of ocular diseases. In this study, we have
`evaluated both local and systemic administration of a
`novel chimeric molecule, VEGF-TRAPR1R2, which com-
`prises portions of the extracellular domain of VEGFR-1
`(flt-1) and VEGFR-2 (KDR), in models of ocular neo-
`vascularization and breakdown of the BRB.
`
`MATERIALS AND METHODS
`VEGF-TRAPR1R2
`Pharmaceuticals,
`(Regeneron
`VEGF-TRAPR1R2
`Tarrytown, NY) is a recombinant fusion protein that
`contains Ig domain 2 of VEGF-R1 and Ig domain 3
`of VEGF-R2 fused to the Fc portion of human IgG1
`
`(Wulff et al., 2002). VEGF-TRAPR1R2 binds VEGF with
`high affinity (kD & 1 pM) and subcutaneous injection
`of 25 mg/kg of VEGF-TRAPR1R2 has been shown to
`effectively neutralize VEGF in mice with VEGF-secret-
`ing tumors (Wong et al., 2001). Recombinant human Fc
`was used as a control protein.
`
`Treatment of mice with laser-induced
`choroidal neovascularization
`Choroidal neovascularization was generated by
`modification of a previously described technique (Tobe
`et al., 1998b). Briefly, 4–5-week-old female C57BL/6J
`mice were anesthetized with ketamine hydrochloride
`(100 mg/kg body weight) and the pupils were dilated
`with 1% tropicamide. Three burns of 532 nm diode laser
`photocoagulation (75 mm spot size, 0.1 sec duration,
`120 mW) were delivered to each retina using the slit
`lamp delivery system of an OcuLight GL Photocoagu-
`lator (Iridex, Mountain View, CA) and a hand held cover
`slide as a contact lens. Burns were performed in the 9, 12,
`and 3 o’clock positions of the posterior pole of the retina.
`Production of a bubble at the time of laser, which
`indicates rupture of Bruch’s membrane, is an important
`factor in obtaining CNV (Tobe et al., 1998b), so only
`burns in which a bubble was produced were included in
`the study. Mice were treated with subcutaneous injec-
`tions of 25 mg/kg of VEGF-TRAPR1R2 or Fc fragment
`1 day prior to laser and on days 2, 5, 8, and 11 after laser.
`At 14 days after laser, the mice were euthanized, serum
`was collected and stored, and eyes were rapidly
`dissected for choroidal flat mounts or frozen in optimum
`cutting temperature embedding compound (OCT; Miles
`Diagnostics, Elkhart, IN).
`Some mice were given intraocular injection of 4.92 mg
`of VEGF-TRAPR1R2 in one eye and 4.92 mg Fc fragment
`in the other eye. Two weeks later, mice were perfused
`with fluorescein-labeled dextran and choroidal neovas-
`cularization was measured.
`
`Quantitative analysis of the amount
`of choroidal neovascularization
`The sizes of CNV lesions were measured in choroidal
`flat mounts (Edelman and Castro, 2000) by an investi-
`gator masked with respect to treatment group. Mice
`used for the flat mount technique were anesthetized and
`perfused with 1 ml of phosphate-buffered saline contain-
`ing 50 mg/ml of fluorescein-labeled dextran (2 106
`average mw, Sigma, St. Louis, MO) as previously des-
`cribed (Tobe et al., 1998a). The eyes were removed and
`fixed for 1 h in 10% phosphate-buffered formalin. The
`cornea and lens were removed and the entire retina was
`carefully dissected from the eyecup. Radial cuts (4–7,
`average 5) were made from the edge to the equator and
`the eyecup was flat mounted in Aquamount with the
`sclera facing down. Flat mounts were examined by
`fluorescence microscopy on an Axioskop microscope
`(Zeiss, Thornwood, NY) and images were digitized using
`a 3 color CCD video camera (IK-TU40A, Toshiba, Tokyo,
`Japan) and a frame grabber. Image-Pro Plus software
`(Media Cybernetics, Silver Spring, MD) was used to
`measure the total area of choroidal neovascularization
`associated with each burn with the operator masked
`with respect to treatment group. Statistical compari-
`sons were made between the size of lesions in mice
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`treated with VEGF-TRAPR1R2 versus those in mice
`treated with Fc fragment by two-tailed t-test. In
`addition, the average size of choroidal neovasculariza-
`tion in each mouse was calculated and plotted against
`the serum level of VEGF-TRAPR1R2 obtained by ELISA.
`In some mice, the eyes were rapidly removed and
`frozen in optimum cutting temperature embedding
`compound (OCT; Miles Diagnostics). Ten mm frozen
`sections were cut through entire lesions and the sections
`were histochemically stained with biotinylated Griffo-
`nia simplicifolia lectin B4 (GSA, Vector Laboratories,
`Burlingame, CA), which selectively binds to vascular
`cells. Slides were incubated in methanol/H2O2 for 10 min
`at 48C, washed with 0.05 M Tris-buffered saline, pH 7.6
`(TBS), and incubated for 30 min in 10% normal porcine
`serum. Slides were incubated 2 h at room temperature
`with biotinylated GSA and after rinsing with 0.05 M
`TBS, they were incubated with avidin coupled to
`peroxidase (Vector Laboratories) for 45 min at room
`temperature. The slides were developed with Histo-
`Mark Red (Kirkegaard and Perry, Cabin John, MD) to
`give a red reaction product and counter stained with
`Contrast Blue (Kirkegaard and Perry).
`
`Transgenic mice with increased expression
`of VEGF in photoreceptors
`Transgenic mice with VEGF driven by the rhodopsin
`promoter develop subretinal neovascularization due to
`expression of VEGF in photoreceptors beginning at
`about P7 (Okamoto et al., 1997). Hemizygous transgene-
`positive mice were given a subcutaneous injection of
`25 mg/kg of VEGF-TRAPR1R2 or Fc fragment at P7, P10,
`P13, P16, and P19. At P21, the mice were sacrificed
`and the amount of subretinal neovascularization was
`quantified as previously described (Tobe et al., 1998a).
`Briefly, mice were anesthetized and perfused with 1 ml
`of phosphate-buffered saline containing 50 mg/ml of flu-
`orescein-labeled dextran (2  106 average mw, Sigma).
`The eyes were removed and fixed for 1 h in 10%
`phosphate-buffered formalin. The cornea and lens were
`removed and the entire retina was carefully dissected
`from the eyecup, radially cut from the edge of the retina
`to the equator in all 4 quadrants, and flat-mounted in
`Aquamount with photoreceptors facing upward. The
`retinas were examined by fluorescence microscopy at
`200x magnification, which provides a narrow depth of
`field so that when focusing on neovascularization on
`the outer surface of the retina, the remainder of the
`retinal vessels are out-of-focus allowing easy delineation
`of the neovascularization. The outer edge of the retina,
`which corresponds to the subretinal space in vivo, is
`easily identified and therefore there is standardization
`of focal plane from slide to slide. Images were digitized
`using a 3 CCD color video camera and a frame grabber.
`Using Image-Pro Plus software, an investigator masked
`with respect to treatment group delineated each of the
`lesions and calculated the total area of neovasculariza-
`tion per retina as previously described (Tobe et al.,
`1998a).
`
`VEGF-induced breakdown of the BRB
`Adult C57BL/6 mice were given a subcutaneous
`injection of 25 mg/kg of VEGF-TRAPR1R2 or Fc and on
`the following day VEGF-induced breakdown of the BRB
`
`was quantified as previously reported (Derevjanik et al.,
`2002). Mice were anesthetized with 25 mg/kg of
`ketamine and 4 mg/kg of xylazine, pupils were dilated
`with 1% tropicamide. Intraocular injections were per-
`formed with a Harvard pump microinjection apparatus
`and pulled glass micropipets (Mori et al., 2001). Each
`micropipet was calibrated to deliver 1 ml of fluid upon
`depression of a foot switch. Under a dissecting micro-
`scope, the sharpened tip of a micropipet was passed
`through the sclera just behind the limbus into the
`vitreous cavity, and the foot switch was depressed
`6 M human vascular endothelial
`injecting 1 ml of 10
`growth factor (VEGF; R&D Systems, Minneapolis, MN).
`Six hours later, retinal vascular permeability was
`measured using [3H]mannitol as a tracer.
`Double transgenic rho/rtTA-TRE/VEGF mice with
`doxycycline-inducible expression of VEGF in photo-
`receptors (Ohno-Matsui et al., 2002) were also used.
`Double transgenics were given a subcutaneous injection
`of 25 mg/kg of VEGF-TRAPR1R2 or Fc fragment of IgG
`and on the following day they were started on 2 mg/ml of
`doxycycline in their drinking water. The next day they
`were given a second subcutaneous injection of 25 mg/kg
`of VEGF-TRAPR1R2 or Fc fragment and after two days,
`retinal vascular permeability was measured.
`
`Measurement of BRB breakdown
`using [3H]mannitol as tracer
`Six hours after intraocular injection of VEGF in wild
`type mice or 2 days after rho/rtTA-TRE/VEGF were
`started on doxycycline, mice were given an intraper-
`itoneal injection of 1 mCi/gram body weight of [3H]man-
`nitol (New England Nuclear, Boston, MA). After 1 h,
`mice were sacrificed and eyes were removed. The cornea
`and lens were removed and the entire retina was
`carefully dissected from the eyecup and placed within
`pre-weighed scintillation vials. The thoracic cavity was
`opened and the left superior lobe of the lung was
`removed and placed in another pre-weighed scintilla-
`tion vial. All liquid was removed from the vials and
`remaining droplets were allowed to evaporate over
`20 min. The vials were weighed and the tissue weights
`were recorded. One ml of NCSII solubilizing solution
`(Amersham, Chicago, IL) was added to each vial and the
`vials were incubated overnight in a 508C water bath.
`The solubilized tissue was brought to room temperature
`and decolorized with 20% benzoyl peroxide in toluene in
`a 508C water bath. The vials were brought to room
`temperature and 5 ml of Cytoscint ES (ICN, Aurora,
`OH) and 30 ml of glacial acetic acid were added. The vials
`were stored for several hours in darkness at 48C to
`eliminate chemoluminescence. Radioactivity was count-
`ed with a Wallac 1409 Liquid Scintillation Counter
`(Gaithersburg, MD).
`
`RESULTS
`Subcutaneous injection of VEGF-TRAPR1R2
`inhibits choroidal neovascularization
`Bruch’s membrane was ruptured at 3 locations in each
`eye by laser photocoagulation in C57BL/6 mice. One day
`prior to laser and on days 2, 5, 8, and 11 after laser, mice
`received subcutaneous injection of 25 mg/kg of VEGF-
`TRAPR1R2 or Fc fragment. Retinal whole mounts from
`fluorescein dextran-perfused mice treated with VEGF-
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`TRAPR1R2 (Fig. 1A,B) had areas of neovascularization
`that were much smaller than those seen in mice treated
`with Fc fragment (Fig. 1C,D). Sections through Bruch’s
`membrane rupture sites in other mice treated with
`VEGF-TRAPR1R2 showed complete or near-complete in-
`hibition of choroidal neovascularization (Fig. 1E,F).
`Mice treated with Fc fragment (Fig. 1G,H) had choroidal
`neovascularization similar to that seen in mice treated
`with vehicle in several other studies (Seo et al., 1999;
`Kwak et al., 2000). Measurement of the area of choroidal
`neovascularization by image analysis confirmed that
`there was significantly less neovascularization in eyes
`treated with VEGF-TRAPR1R2 compared to those trea-
`ted with Fc fragment (Fig. 1I). The level of VEGF-
`
`TRAPR1R2 was measured in plasma obtained from each
`of the mice at the time of sacrifice. Each of the mice that
`had been injected with Fc fragment had no detectable
`VEGF-TRAPR1R2 in its plasma, while mice that had
`been injected with VEGF-TRAPR1R2 had plasma levels
`ranging from 57 to 205 mg/ml. All of the plasma levels of
`VEGF-TRAPR1R2 between 57 and 205 mg/ml were
`associated with strong inhibition of choroidal neovascu-
`larization (Fig. 1J).
`Immediately after laser, some mice were given
`intraocular injection of VEGF-TRAPR1R2 or Fc fragment
`of IgG. Two weeks later, mice were perfused with
`fluorescein-labeled dextran and choroidal neovascular-
`ization was measured. Mice that received intraocular
`
`Fig. 1. Subcutaneous VEGF-TRAPR1R2 suppresses choroidal neovas-
`cularization at sites of rupture of Bruch’s membrane. Adult C57BL/6
`mice were had rupture of Bruch’s membrane by laser photocoagula-
`tion in 3 locations in each eye. Prior to laser and on days 2, 5, 8, and
`11 after laser, mice received subcutaneous injection of 25 mg/kg of
`VEGF-TRAPR1R2 or Fc fragment of IgG. Parts A and B show small
`areas of neovascularization (surrounded by arrows) in retinal whole
`mounts from two fluorescein dextran-perfused mice treated with
`VEGF-TRAPR1R2. Griffonia simplicifolia (GSA) lectin-stained sections
`in two other mice treated with VEGF-TRAPR1R2 show minimal
`choroidal neovascularization (E- none visible and F- between arrows).
`Parts C and D show large areas of neovascularization (surrounded by
`
`arrows) in choroidal flat mounts from two Fc fragment-treated mice
`and GSA-stained sections from two other mice treated with Fc
`fragment show prominent areas of neovascularization (G and H,
`between arrows). Measurement by image analysis of the area of
`neovascularization on choroidal flat mounts (I) showed an average
`area that was significantly smaller ( P < 0.0001 by Student’s two-tailed
`t-test) in VEGF-TRAPR1R2-treated mice (20 eyes, 52 rupture sites)
`compared to Fc-treated mice (20 eyes, 57 rupture sites). Plasma levels
`of VEGF at the time of sacrifice determined by ELISA plotted against
`the average area of choroidal neovascularization per mouse showed
`marked suppression of neovascularization at all plasma levels
`between 50 and 200 mg/ml (J). Bar ¼ 100 mm.
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`Fig. 2. A single intravitreous injection of VEGF-TRAPR1R2 sup-
`presses choroidal neovascularization at Bruch’s membrane rupture
`sites. Immediately after laser, C57BL/6 mice were given intraocular
`injection of 4.92 mg of VEGF-TRAPR1R2 in one eye and 4.92 mg of Fc
`fragment in the other eye. Two weeks later, mice were perfused with
`fluorescein-labeled dextran and choroidal neovascularization was
`measured. A and B: Large areas of neovascularization (surrounded
`by arrows) are seen in flat mounts from two separate mice treated with
`
`intravitreous injection of Fc fragment. C and D: Small areas of
`neovascularization (surrounded by arrows) are seen in two sepa-
`rate mice given a single intravitreous injection of VEGF-TRAPR1R2.
`E: The area of choroidal neovascularization measured by image
`analysis was significantly less (P < 0.0001; Student’s two-tailed t-test)
`in VEGF-TRAPR1R2-treated eyes (19 eyes, 54 rupture sites) compared
`to Fc-treated eyes (19 eyes, 44 rupture sites). Bar ¼ 100 mm
`
`injection of Fc fragment had larger areas of choroidal
`neovascularization (Fig. 2A,B) than those seen in
`mice that received a single intraocular injection of
`VEGF-TRAPR1R2 (Fig. 2C,D). There was a statistically
`significant reduction in the mean area of neovascular-
`ization in VEGF-TRAPR1R2-injected eyes compared to
`Fc fragment-injected eyes (Fig. 2E).
`
`VEGF-TRAPR1R2 inhibits subretinal
`neovascularization in Rho/VEGF
`transgenic mice
`
`Rho/VEGF transgenic mice express VEGF in photo-
`receptors starting about postnatal day (P) 7 resulting
`in extensive subretinal neovascularization by P21
`
`Fig. 3. Subcutaneous VEGF-TRAPR1R2 inhibits subretinal neovas-
`cularization in rho/VEGF transgenic mice. Rho/VEGF transgenic mice
`begin to express VEGF in photoreceptors about postnatal day (P) 7.
`At P7, mice were divided into two groups and treated with 25 mg/kg of
`VEGF-TRAPR1R2 (9 mice, 17 eyes) or Fc fragment (10 mice, 19 eyes) on
`P7, P10, P13, P16, and P19, and on P21, the mice were anesthetized
`and perfused with fluorescein-labeled dextran. Retinal whole mounts
`
`from mice treated with VEGF-TRAPR1R2 showed few areas of
`neovascularization (A and B, arrows), while there were numerous
`clumps of new vessels in the subretinal space of mice that had been
`treated with Fc fragment (C and D, arrows). Measurement of the total
`area of neovascularization per retina by image analysis showed
`significantly less neovascularization in VEGF-TRAPR1R2-treated
`mice, compared to those treated with Fc fragment (E). Bar ¼ 100 mm.
`
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`(Okamoto et al., 1997; Tobe et al., 1998a). Rho/VEGF
`mice received subcutaneous injection of 25 mg/kg of
`VEGF-TRAPR1R2 or Fc fragment of IgG on P7, P10, P13,
`P16, and P19, and on P21, they were perfused with
`fluorescein-labeled dextran. Mice treated with VEGF-
`TRAPR1R2 had very few clumps of neovascularization
`(Fig. 3A,B, arrows), while there were numerous clumps
`of new vessels in the subretinal space of mice that had
`been treated with Fc fragment of IgG (Fig. 3C,D,
`arrows). Image analysis showed that mice treated with
`VEGF-TRAPR1R2 had an average area of neovascular-
`ization per retina that was significantly smaller total
`area than mice treated with Fc fragment (Fig. 3E).
`
`VEGF-TRAPR1R2 inhibits VEGF-induced
`breakdown of the BRB
`Adult C57BL/6 mice were given a subcutaneous
`injection of 25 mg/kg of VEGF-TRAPR1R2 or Fc fragment
`and on the following day received an intravitreous
`6 M VEGF. Six hours later, retinal
`injection of 1 mg of 10
`vascular permeability was measured using [3H]manni-
`tol as a tracer. Mice treated with VEGF-TRAPR1R2 had a
`significantly smaller retina to lung leakage ratio than
`mice treated with Fc fragment of IgG indicating less
`breakdown of the BRB (Fig. 4A).
`We have previously produced and characterized
`double transgenic mice with doxycycline-inducible
`expression of VEGF in the retina (Ohno-Matsui et al.,
`2002). Double transgenics were given a subcutaneous
`injection of 25 mg/kg of VEGF-TRAPR1R2 or Fc fragment
`and on the following day they were started on 2 mg/ml of
`doxycycline in their drinking water. Two days later, they
`were given a second subcutaneous injection of 25 mg/kg
`of VEGF-TRAPR1R2 or Fc fragment and then the next
`day retinal vascular permeability was measured with
`[3H]mannitol. Double transgenic mice treated with
`VEGF-TRAPR1R2 had a significant reduction in the
`retina to lung leakage ratio compared to mice treated
`with Fc fragment (Fig. 4B).
`
`DISCUSSION
`Retinal ischemia is the underlying cause of retinal
`neovascularization. Since VEGF and VEGFR1 are
`upregulated in ischemic tissue (Forsythe et al., 1996;
`Gerber et al., 1997; Iyer et al., 1998), it is not surprising
`that VEGF plays a central role in the pathogenesis of
`retinal neovascularization. The pathogenesis of chor-
`oidal neovascularization is poorly understood. Choroi-
`dal blood flow is decreased in patients with AMD
`(Grunwald et al., 1998; Ross and Barofsky, 1998), but
`it is not known if this is sufficient to cause hypoxia. Also,
`it is unlikely that hypoxia is present in other disease
`processes, such as ocular histoplasmosis or degenerative
`myopia, in which choroidal neovascularization occurs in
`young patients. Since ischemia has not been implicated
`in the pathogenesis of choroidal neovascularization,
`this piece of evidence that made VEGF a prime suspect
`for retinal neovascularization is lacking for choroidal
`neovascularization. On the other hand, surgically re-
`moved choroidal neovascular membranes show im-
`munohistochemical staining for VEGF (Amin et al.,
`1994; Frank et al., 1996; Kvanta et al., 1996; Lopez et al.,
`1996) and there is increased VEGF mRNA in experi-
`mentally induced choroidal neovascularization (Ogata
`
`Fig. 4. Subcutaneous injections of VEGF-TRAPR1R2 suppress VEGF-
`induced breakdown of the BRB. Adult C57BL/6 mice were given a
`subcutaneous injection of 25 mg/kg of VEGF-TRAPR1R2 or Fc fragment
`and on the following day received an intravitreous injection of 1 mg of
`6 M VEGF. Six hours later, retinal vascular permeability was
`10
`measured using [3H]mannitol as a tracer. Mice treated with VEGF-
`TRAPR1R2 (9 mice, 18 eyes) had a significantly smaller retina to lung
`leakage ratio (RLLR) than mice treated with Fc fragment (9 mice,
`18 eyes) indicating less breakdown of the BRB (A). Double transgenic
`rtTA/rho-TRE/VEGF mice with doxycycline-inducible expression of
`VEGF in the retina were given a subcutaneous injection of 25 mg/kg of
`VEGF-TRAPR1R2 (10 mice, 20 eyes) or Fc fragment (10 mice, 20 eyes)
`and on the following day they were started on 2 mg/ml of doxycycline
`in their drinking water. Two days later, they were given a second
`subcutaneous injection of 25 mg/kg of VEGF-TRAPR1R2 or Fc fragment
`and then the next day retinal vascular permeability was measured
`with [3H]mannitol as described in Materials and Methods. Double
`transgenic mice treated with VEGF-TRAPR1R2 had a significant
`reduction in the retina to lung leakage ratio compared to mice treated
`with Fc fragment (B).
`
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`VEGF-TRAP AND OCULAR NV
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`247
`
`et al., 1996; Yi et al., 1997). Using a combination of
`kinase inhibitors, we previously demonstrated that
`VEGF signaling is necessary for development of chor-
`oidal neovascularization after laser-induced rupture of
`Bruch’s membrane (Kwak et al., 2000). In the present
`study, using VEGF-TRAPR1R2, a completely different
`type of VEGF inhibitor that is highly specific, we have
`confirmed that VEGF plays a prominent role in the
`development of choroidal neovascularization.
`Systemic administration of VEGF-TRAPR1R2 also
`markedly decreased neovascularization in rho/VEGF
`transgenic mice and reduced VEGF-induced breakdown
`of the BRB. Systemic administration of an earlier
`version of the VEGF-Trap also has been shown to reduce
`elevated ICAM-1 and eNOS levels, inhibit leukostasis,
`and normalize vascular permeability in the retinas of
`diabetic rodents (Qaum et al., 2001; Joussen et al., 2002;
`Poulaki et al., 2002). Thus, in model disease settings
`similar to diabetic retinopathy in humans, circulating
`VEGF-Traps penetrate into the retina and exert a
`strong therapeutic effect. The angiogenic stimulus is
`sustained in rho/VEGF mice, and subcutaneous injec-
`tions of VEGF-TRAPR1R2 every third day provided
`intraocular levels sufficient to neutralize this sustained
`stimulus. These data suggest that VEGF-TRAPR1R2
`deserves consideration as a potential treatment for two
`complications of diabetic retinopathy, retinal neovascu-
`larization and macular edema.
`The effects of long-term systemic inhibition of VEGF
`are unknown. While there are theoretical reasons why
`this could be problematic, VEGF inhibitors have been
`tested as adjuncts to chemotherapy in cancer trials, and
`there have not been reports of severe problems clearly
`linked to blockade of VEGF. Should systemic inhibi-
`tion of VEGF prove problematic, there is an alter-
`native, because we have shown that, as is the case for
`other anti-VEGF approaches (EyeTech Study Group,
`2002; Kryzstolik et al., 2002), local administration of
`VEGF-TRAPR1R2 by intravitreous injection is a viable
`alternative. A single intravitreous injection of VEGF-
`TRAPR1R2 markedly suppressed the development of
`choroidal neovascularization over the course of two
`weeks.
`This study suggests that VEGF-TRAPR1R2 has poten-
`tial as a therapeutic agent for several VEGF-related
`retinal and choroidal diseases. Clinical trials are ne-
`eded to assess the effect of subcutaneously administered
`VEGF-TRAPR1R2 in patients with retinal neovascular-
`ization and/or macular edema due to ischemic retino-
`pathies including diabetic retinopathy and retinal vein
`occlusions, and in patients with choroidal neovascular-
`ization. Concurrently, additional preclinical studies
`should explore modes of local delivery to the eye that
`can be used adjunctively or as an alternative to systemic
`administration.
`
`ACKNOWLEDGMENTS
`The Research to Prevent Blindness grant was
`awarded to P.A.C. as Lew R. Wasserman Merit Awards.
`
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