September 2000
Volume 41, Issue 10
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Retinal Cell Biology  |   September 2000
VEGF Is Major Stimulator in Model of Choroidal Neovascularization
Author Affiliations
  • Naoyuki Okamoto
    From the Departments of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore Maryland; and the Division of Oncology Research, Pharmaceutical Division, Research and Development, Novartis Pharmaceuticals, Basel, Switzerland.
  • Jeanette M. Wood
    From the Departments of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore Maryland; and the Division of Oncology Research, Pharmaceutical Division, Research and Development, Novartis Pharmaceuticals, Basel, Switzerland.
  • Peter A. Campochiaro
    From the Departments of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore Maryland; and the Division of Oncology Research, Pharmaceutical Division, Research and Development, Novartis Pharmaceuticals, Basel, Switzerland.
Investigative Ophthalmology & Visual Science September 2000, Vol.41, 3158-3164. doi:
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      Nohoon Kwak, Naoyuki Okamoto, Jeanette M. Wood, Peter A. Campochiaro; VEGF Is Major Stimulator in Model of Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2000;41(10):3158-3164.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Vascular endothelial growth factor (VEGF) is upregulated by hypoxia and is a major stimulatory factor for retinal neovascularization in ischemic retinopathies such as diabetic retinopathy. This study sought to determine if VEGF is a stimulatory factor in a murine model of choroidal neovascularization (CNV).

methods. Mice with laser-induced ruptures in Bruch’s membrane were treated with vehicle alone; a drug that inhibits both VEGF and platelet-derived growth factor (PDGF) receptor kinases; a drug that inhibits PDGF, but not VEGF receptor kinase; or genistein, a nonspecific kinase inhibitor. After two weeks, CNV was quantified and compared. results. Blockade of phosphorylation by VEGF and PDGF receptors caused dramatic, almost complete inhibition of CNV. Genistein also had an inhibitory effect, but less so than the VEGF/PDGF receptor blocker. Blockade of phosphorylation by PDGF receptors, but not VEGF receptors, had no significant effect on cnv. conclusions. These data and our previous study, which demonstrated that a kinase inhibitor that blocks VEGF and PDGF receptors and several isoforms of protein kinase C causing dramatic inhibition of CNV, suggest that VEGF signaling plays a critical role in the development of CNV in this model. If safety is established, the effect of inhibiting VEGF receptor kinase activity should be investigated in patients with CNV.

Ocular neovascularization is responsible for the majority of cases of new blindness in developed countries every year. Retinal and iris neovascularization occur in the same disease processes, including diabetic retinopathy, the most common cause of severe loss of vision in young people. 1 2 Choroidal neovascularization (CNV) occurs in diseases in which there are abnormalities of Bruch’s membrane and the retinal pigmented epithelium (RPE). The most common disease of this type is age-related macular degeneration (AMD), the most prevalent cause of severe loss of vision in patients more than 60 years of age. 3  
The pathogenesis of retinal neovascularization is much better understood than the pathogenesis of CNV. Occlusion of retinal vessels leading to ischemia or hypoxia is a common feature of diseases in which retinal neovascularization occurs. 4 5 6 Another well-established fact is that one of the stimulatory factors involved is vascular endothelial growth factor (VEGF). It is unlikely to be the only stimulatory factor, because insulin-like growth factor-1 may also participate, 7 but there is strong evidence indicating that VEGF plays a central role. It is upregulated by hypoxia, 8 9 10 and its levels are increased in the retina and vitreous of patients 11 12 13 14 or laboratory animals 15 16 with ischemic retinopathies. Increased expression of VEGF in retinal photoreceptors of transgenic mice stimulates neovascularization within the retina, 17 18 and VEGF antagonists partially inhibit retinal or iris neovascularization in animal models. 19 20 21  
It is not known whether VEGF is also a stimulatory factor for CNV. Unlike retinal neovascularization, it is not clear that hypoxia, a principal cause of upregulation of VEGF, 10 plays a role in CNV. There is some suggestion that choroidal blood flow may be altered in patients with AMD, 22 23 but it is not known whether this is sufficient to cause hypoxia. Also, it is unlikely that hypoxia is present in other disease processes, such as ocular histoplasmosis, in which CNV occurs in young patients. However, increased expression of VEGF has been demonstrated in CNV removed from patients 24 25 26 27 and in experimentally induced CNV. 28 29 But increased expression in association with CNV is not unique to VEGF, because similar data have been obtained for fibroblast growth factor (FGF)-2. 24 25 28 29 . Also, choroidal vessels differ from retinal vessels in several respects, and it is not clear that they are responsive to VEGF. In transgenic mice that express VEGF in photoreceptors, new vessel sprouts develop from retinal vessels, but not from choroidal vessels. 17 Therefore, it is not known whether VEGF is a stimulatory factor for CNV. 
Growth factors such as VEGF act by binding to specific receptors and inducing them to form receptor dimers. The intracellular portion of receptors have kinase domains that phosphorylate other proteins, and each component of a ligand-bound receptor pair phosphorylates its partner, a process known as receptor autophosphorylation. Autophosphorylation increases the affinity of receptor kinases for second messengers that are phosphorylated to propagate the signal that ultimately results in altered gene expression. Therefore, the kinase activity of a receptor is absolutely necessary for its signaling capability and for any biologic process the receptor mediates. 
Our laboratory has recently demonstrated striking inhibition of CNV in a murine model using a selective kinase inhibitor that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC). 30 In this study, we have used other kinase inhibitors with overlapping, but different activities to explore the signaling pathways involved in the development of CNV. 
Materials and Methods
Generation of CNV in Mice
C57BL/6J mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. CNV was generated by laser-induced rupture of Bruch’s membrane, as previously described. 31 Briefly, 4- to 5-week-old male C57BL/6J mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight) and the pupils were dilated with 1% tropicamide. One burn of krypton laser photocoagulation (100-μm spot size, 0.1-second duration, 150 mW) was delivered to the 9, 12, and 3 o’clock positions of the posterior pole of the retina in each eye using the slit lamp delivery system of a photocoagulator (model 920; Coherent, Palo Alto, CA) and a handheld cover slide as a contact lens. Production of a bubble at the time of laser exposure, which indicates rupture of Bruch’s membrane, is an important factor in inducing CNV, 31 and therefore only mice in which a bubble was produced for all six burns were included in the study. 
Treatment of Mice with Kinase Inhibitors by Gavage
Forty mice (10 in each group) were randomly assigned to treatment by gavage with vehicle alone or with one of three kinase inhibitors. The first group was treated with vehicle, and the second group was treated with 50 mg/kg once a day of PTK787/ZK 222584 (PTK787), a selective inhibitor of VEGF receptor kinases and other class III kinases, but not kinases from other classes. 32 33 A third group was treated once a day with the molar equivalent (120 micromoles/kg; 46 mg/kg) of a selective inhibitor of PDGF receptor kinases. 34 35 The fourth group was treated once a day with 120 micromoles/kg (32 mg/kg) of genistein, a nonselective tyrosine kinase inhibitor, that also has some effects unrelated to tyrosine phosphorylation. 36 Mice were treated by gavage on the day of laser and then once a day for 2 weeks when they were killed and evaluated. 
Staining of CNV with a Vascular Cell–Specific Marker
After 14 days of treatment, the mice were killed with an overdose of sodium pentobarbital, and their eyes were rapidly removed and frozen in optimal cutting temperature embedding compound (OCT; Miles, Elkhart, IN). Frozen serial sections (10 μm) were cut through the entire extent of each of the three CNV lesions associated with laser sites in each eye. Sections were histochemically stained as previously described 37 with biotinylated Griffonia simplicifolia lectin B4 (GSA; Vector, Burlingame, CA), which selectively binds to vascular cells. Slides were incubated in 4% paraformaldehyde for 30 minutes, washed with 0.05 M Tris buffer, (TB; pH 7.4), incubated in methanol-H2O2 for 10 minutes at 4°C, washed with 0.05 M TB, and incubated for 30 minutes in 10% normal swine serum. Slides were rinsed with 0.05 M TB and incubated 2 hours at 37°C with 1:20 biotinylated lectin. After they were rinsed with 0.05 M TB, slides were incubated with undiluted streptavidin-phosphatase (Kirkegaard & Perry, Cabin John, MD) for 30 minutes at room temperature. After a 10-minute wash in 0.05 M TB (pH 7.6) slides were developed with staining (HistoMark Red 38 ; Kirkegaard & Perry). Some slides were counterstained (Contrast Blue; Kirkegaard & Perry). 
Quantitative Analysis of the Amount of CNV per Lesion
To perform quantitative assessments, GSA-stained sections were examined at ×400 with an (Axioskop; Carl Zeiss, Thornwood, NY) microscope with the observer masked to treatment group. Images were digitized using a three CCD color video camera and a frame grabber. GSA-stained blood vessels were delineated and the area in the subretinal space measured by computer with image analysis software (Image-Pro Plus software; Media Cybernetics, Silver Spring, MD). For each CNV lesion, area measurements were made for all sections on which some of the lesion appeared and added together to give the integrated area measurement. Only CNV lesions in which good sections were obtained through the entire extent of CNV, so that a valid area measurement could be made on each, were included in the analysis. Lesions were excluded based solely on the inability to obtain an accurate measurement because of poor quality of some sections from that particular CNV lesion with the observer masked with regard to treatment group, so that there was no possibility of a bias with regard to lesion exclusion. After unmasking, it was found that precise measurements had been obtained on the following number of lesions in each treatment group: 52 controls, 56 PTK787, 52 CGP 53716, and 52 genistein. There were 60 CNV lesions in each group, and therefore there were 4 that were unmeasurable in the PTK787 group and 8 in each of the other three groups. 
Determination of the Effect of Kinase Inhibitors on Normal Retinal Vessels
Adult C57BL/6J mice (eight mice in each group) were treated by gavage with vehicle or vehicle containing 120 micromoles/kg per day of one of the kinase inhibitors. After 2 weeks, mice were killed with an overdose of pentobarbital sodium, and their eyes were rapidly removed and frozen in OCT. Serial sections (10 μm) were cut entirely through each eye and stained with GSA. The retinal vessel area was determined by image analysis on every 10th section, and the mean was calculated for each eye as previously described. 37  
Statistical Analysis
Because more than one CNV lesion was created in each mouse, repeated measures analysis of variance was used to compare differences in the area of CNV lesions among mice, and after adjusting for differences among mice, comparison of differences in the area of CNV lesions among treatment groups was performed. To provide a graphic demonstration that portrays the differences among mice within treatment groups as well as the differences among treatment groups, box plots were made for each group showing the median log (CNV area), the 25% to 75% range, and the highest and lowest value. The same analysis was performed to make statistical comparisons of retinal vascular area for the toxicity study. 
Results
Laser-Induced CNV
Two weeks after laser photocoagulation, histopathologic evaluation showed a discontinuity in Bruch’s membrane in the area of each laser burn in all mice. Control mice that were treated with aqueous buffer (phosphate-buffered saline [PBS]) or buffer containing 1% dimethyl sulfoxide, showed large areas of CNV at each site of laser-induced rupture of Bruch’s membrane (Fig. 1A , 1E) . Previous characterization of this model has demonstrated the origin of the neovascularization to be from choroidal vessels. 31 There was proliferation of RPE cells along the margin of the new vessels. The overlying lectin-stained retinal blood vessels were normal. 
Inhibition of CNV Due to Blockade of VEGF and PDGF
Selective kinase inhibitors were used to explore the signaling pathways involved in the development of CNV. PTK787 is a good inhibitor of phosphorylation by human VEGF receptor 2 and its mouse homologue, Flk-1 (50% inhibitory concentrations [IC50s]: 0.1 and 0.27 μM, respectively). 32 33 It is a less potent inhibitor of VEGF receptor 1 (IC50: 0.49 μM) and also blocks the related tyrosine kinases PDGF β-receptor, c-Kit (the receptor for stem cell factor), and cfms (the receptor for macrophage colony stimulating factor-1; IC50s: 0.2, 0.38, and 1.2 μM, respectively). In concentrations up to 10μ M, PTK787 does not inhibit kinases from other families including FGFR1, c-Met, Tie2, epithelial growth factor receptor (EGFR), c-Src, v-Abl, or PKC-α. 
Mice treated with the dihydrochloride salt of PTK787 dissolved in PBS, showed a dramatic decrease in the size of CNV associated with laser-induced rupture of Bruch’s membrane (Figs. 1B 1F) . In many instances, there was no identifiable lectin-stained neovascular tissue throughout the entire burn, but some burns contained regions in which there were thin disks of lectin-stained tissue. There was mild proliferation of RPE cells. Despite the marked decrease in CNV in the eyes of treated mice, the overlying retinal vessels appeared normal. This is best seen in sections with no counterstain (Fig. 1B)
Measurement by image analysis of the integrated area of lectin staining per lesion showed a dramatic decrease in mice treated with PTK787 (0.0134638 ± 0.001799 mm2) compared with lesions in mice treated with vehicle (PBS) alone (0.0627643 ± 0.008704 mm2). This difference was highly statistically significant (P < 0.0001 by repeated measures analysis of variance [ANOVA]; Fig. 2 ). 
Lack of Inhibition of CNV Due to Blockade of PDGF Recepter Kinase Alone
CGP 53716 is a selective inhibitor of the kinase activity of PDGFβ -receptors (IC50: 0.1 μM) and v-Abl (IC50: 0.6 μM) and does not inhibit phosphorylation by other kinases, including VEGF receptors or PKCs in concentrations greater than 20 μM. 34 35 Mice treated with CGP 53716, showed large areas of CNV similar to those seen in control mice (Figs. 1C 1G) . Measurement of CNV lesion size by image analysis demonstrated that there was no significant difference in the integrated area of lectin staining per lesion in CGP 53716–treated mice compared with vehicle-treated mice (P = 0.56 by repeated measures ANOVA). 
Parital Inhibition of CNV by Genistein
Mice treated with genistein (Figs. 1D 1H) , a nonspecific kinase inhibitor, showed areas of CNV that appeared intermediate in size between control mice and those treated with PTK787. Image analysis confirmed that this was the case. Mice treated with genistein had lesions with an integrated area of lectin staining that was significantly smaller than that in control mice (P < 0.0001) but greater than that in PTK787-treated mice (P < 0.0001; Fig. 2 ). Expressed as a mathematical relationship, the area of CNV in the four treatment groups is vehicle control = CGP 53716 > genistein > PTK787. 
Kinase Inhibitors Lack Identifiable Toxic Effect on Normal Retinal Blood Vessels
To investigate for toxic effects on retinal vessels, mice that did not receive any laser photocoagulation were treated with PTK787, CGP 53716, or genistein for 2 weeks. None of the drugs caused changes in morphology of retinal vessels (notshown), and there were no significant differences in retinal vascular area per section compared with control mice (Fig. 3)
Discussion
Identification of stimulatory factors for a pathologic process makes it possible to design drug treatments that are likely to inhibit the process with minimal unwanted side effects. There are several lines of evidence suggesting that VEGF plays a major role in the stimulation of retinal neovascularization and therefore many strategies for inhibiting VEGF signaling are under study as potential treatments. 19 20 21 Less is known concerning possible stimulatory factors for CNV, but it has been noted that expression of FGF2 and VEGF is increased in association with the development of CNV. 24 25 26 27 28 29 We have recently demonstrated that FGF2 is not necessary for the development of CNV, suggesting that it does not play a central role. 31 In this study and another recent study, 30 we used kinase inhibitors with overlapping but different activities, to demonstrate that inhibition of VEGF receptor tyrosine kinase activity dramatically inhibits CNV. This indicates that VEGF is a stimulator for CNV in this model. 
Our experimental paradigm involved initiation of drug treatment right after laser-induced rupture of Bruch’s membrane. Therefore, it is relevant to prevention of CNV, but not necessarily regression of already established CNV. Also, although the murine laser-induced model does not exactly mimic all aspects of CNV that occurs in association with AMD, it shares two critical features: The new vessels originate from choroidal vessels, and there are abnormalities in Bruch’s membrane. Laser-induced disruption of Bruch’s membrane in humans is also associated with CNV. 39 40 41 Therefore, our data provide suggestive, although not definitive, evidence that VEGF is a stimulatory factor for CNV in humans. 
What is the source of VEGF in the setting of CNV? Studies of human pathologic specimens 25 26 27 as well as a study in a laser-induced experimental model, 29 suggest that RPE cells are the source of VEGF, and it is well-established that cultured RPE cells produce VEGF. 42 But there is no evidence suggesting that RPE cells are hypoxic in patients in whom CNV develops. Hypoxia of the RPE seems particularly unlikely in patients with ocular histoplasmosis, angioid streaks, pathologic myopia, or choroidal rupture, all of which are conditions in which there is a high incidence of CNV. Features that these conditions share with each other and with AMD, are disruption of Bruch’s membrane and/or other abnormalities of the extracellular matrix of the RPE and Bruch’s membrane. We have recently demonstrated that exposure of cultured RPE cells to certain extracellular matrix proteins that are not normally part of the microenvironment of RPE cells, particularly thrombospondin 1, can increase secretion of VEGF. 43 Therefore, alterations in Bruch’s membrane accompanied by changes in the extracellular matrix of RPE cells may contribute to increased production of VEGF in patients with CNV. 
Transgenic mice that express high levels of VEGF in photoreceptors, show neovascularization originating from retinal vessels, but not choroidal vessels. 17 Perhaps normal RPE cells prevent retina-derived VEGF from getting to choroidal vessels. Another possibility is that normal RPE cells and/or Bruch’s membrane provide a physical or biochemical barrier to vascular invasion from the choroid. Mice or rats with photoreceptor degeneration frequently have neovascularization that extends from retinal or choroidal vessels into the RPE, 44 and we have demonstrated that photoreceptor degeneration results in increased expression of VEGF in RPE cells. 45 Taken together, these various pieces of evidence suggest that exposure of RPE cells to certain extracellular matrix components results in increased production of VEGF and that unlike retina-derived VEGF, RPE-derived VEGF gains access to choroidal vessels and can stimulate CNV. 
The results of the present study suggest that VEGF plays an important role in the stimulation of CNV, but they do not rule out the participation of other stimulatory factors. However, blockade of VEGF signaling dramatically, and in many mice completely, inhibits CNV. Therefore, even if there are other contributing stimulatory factors, it may be possible to effectively prevent CNV in patients by concentrating solely on inhibition of VEGF signaling. This also appears to be an effective strategy for retinal neovascularization, because blockage of VEGF signaling completely inhibits retinal neovascularization in murine oxygen-induced ischemic retinopathy. 30 46 These data suggest that if safety is established, these drugs should be tested for their ability to inhibit ocular neovascularization in patients. 
 
Figure 1.
 
Effect of kinase inhibitors with overlapping activity on CNV in a murine model. Mice with laser-induced rupture of Bruch’s membrane were treated orally by gavage for 2 weeks with vehicle alone (A, E) or with vehicle containing 120 micromoles/kg PTK787 (B, F), CGP 57148 (C, G), or genistein (D, H). Retinal sections from two mice with each treatment were histochemically stained with GSA, which selectively stains vascular cells. Sections were either not counterstained, to provide optimal visualization of retinal vessels (A through D), or were counterstained with contrast blue to allow visualization of retinal neurons (E through H). Arrows: Horizontal extent of the CNV. There were large areas of choroidal neovascularization in mice treated with vehicle alone (A, E) or CGP 57148, the inhibitor of PDGF, but not VEGF receptor kinase activity (C, G). In contrast, mice treated with PTK787, an inhibitor of both VEGF and PDGF receptor kinase activity, had almost no choroidal neovascularization (B, F). Mice treated with genistein, a nonspecific kinase inhibitor, had an intermediate amount of choroidal neovascularization.
Figure 1.
 
Effect of kinase inhibitors with overlapping activity on CNV in a murine model. Mice with laser-induced rupture of Bruch’s membrane were treated orally by gavage for 2 weeks with vehicle alone (A, E) or with vehicle containing 120 micromoles/kg PTK787 (B, F), CGP 57148 (C, G), or genistein (D, H). Retinal sections from two mice with each treatment were histochemically stained with GSA, which selectively stains vascular cells. Sections were either not counterstained, to provide optimal visualization of retinal vessels (A through D), or were counterstained with contrast blue to allow visualization of retinal neurons (E through H). Arrows: Horizontal extent of the CNV. There were large areas of choroidal neovascularization in mice treated with vehicle alone (A, E) or CGP 57148, the inhibitor of PDGF, but not VEGF receptor kinase activity (C, G). In contrast, mice treated with PTK787, an inhibitor of both VEGF and PDGF receptor kinase activity, had almost no choroidal neovascularization (B, F). Mice treated with genistein, a nonspecific kinase inhibitor, had an intermediate amount of choroidal neovascularization.
Figure 2.
 
Integrated area of CNV and box plot of the log values of the integrated area of CNV in control mice and mice treated with PTK787, CGP 53716, or genistein. Mice with laser-induced rupture of Bruch’s membrane were treated orally by gavage with vehicle or 120 micromoles/kg of one of the drugs for 2 weeks. Serial sections through the entire extent of each lesion were stained with GSA, and the area of CNV on each section was measured by image analysis. All the area measurements for each lesion were added together to generate the integrated area of CNV. Mean ± SEM is plotted on the left in the bar graph (n for each treatment group: 52 for control; 52, genistein; 56, PTK787; and 52, CGP 53716). *P < 0.001 by repeated-measures ANOVA for difference from control. The log(integrated area of CNV) for each group is shown in a box plot on the right in which the central line is the median, the box contains all values between the 25th and 75th percentiles, and the lines show the highest and lowest value in each group, except for the control and CGP 53716 group in which the highest are shown by circles. Statistical comparisons by repeated measures analysis of variance showed P < 0.001 for differences between control and PTK787, control and genistein, and PTK787 and genistein. P = 0.56 for difference between control and CGP 53716.
Figure 2.
 
Integrated area of CNV and box plot of the log values of the integrated area of CNV in control mice and mice treated with PTK787, CGP 53716, or genistein. Mice with laser-induced rupture of Bruch’s membrane were treated orally by gavage with vehicle or 120 micromoles/kg of one of the drugs for 2 weeks. Serial sections through the entire extent of each lesion were stained with GSA, and the area of CNV on each section was measured by image analysis. All the area measurements for each lesion were added together to generate the integrated area of CNV. Mean ± SEM is plotted on the left in the bar graph (n for each treatment group: 52 for control; 52, genistein; 56, PTK787; and 52, CGP 53716). *P < 0.001 by repeated-measures ANOVA for difference from control. The log(integrated area of CNV) for each group is shown in a box plot on the right in which the central line is the median, the box contains all values between the 25th and 75th percentiles, and the lines show the highest and lowest value in each group, except for the control and CGP 53716 group in which the highest are shown by circles. Statistical comparisons by repeated measures analysis of variance showed P < 0.001 for differences between control and PTK787, control and genistein, and PTK787 and genistein. P = 0.56 for difference between control and CGP 53716.
Figure 3.
 
Log value of retinal vessel area per section in control mice and mice treated for 2 weeks with 120 micromoles/kg of PTK787, CGP 53716, or genistein. Serial sections were cut through entire eyes and stained with GSA. The retinal vessel area was determined by image analysis on every 10th section, and the mean was calculated for each eye. The box plot and symbols are as described for Figure 2 . Statistical comparisons were made by repeated-measures ANOVA, and there were no significant differences between any members of the group.
Figure 3.
 
Log value of retinal vessel area per section in control mice and mice treated for 2 weeks with 120 micromoles/kg of PTK787, CGP 53716, or genistein. Serial sections were cut through entire eyes and stained with GSA. The retinal vessel area was determined by image analysis on every 10th section, and the mean was calculated for each eye. The box plot and symbols are as described for Figure 2 . Statistical comparisons were made by repeated-measures ANOVA, and there were no significant differences between any members of the group.
The authors thank Michele Melia of the Wilmer Institute Core statistics center for performing the statistical analysis. 
Kahn H, Hiller R. Blindness caused by diabetic retinopathy. Am J Ophthalmol. 1974;78:58–67. [CrossRef] [PubMed]
Klein R, Klein B. Vision disorders in diabetes. National Diabetes Data Group eds. Diabetes in America. 1995; 2nd ed. 293–330. National Institutes of Health Bethesda, MD.
Macular Photocoagulation Study Group. Argon laser photocoagulation for neovascular maculopathy: five year results from randomized clinical trials. Arch Ophthalmol. 1991;109:1109–1114. [CrossRef] [PubMed]
Michaelson I. The mode of development of the vascular system of the retina with some observations on its significance for certain retinal diseases. Trans Ophthalmol Soc UK. 1948;68:137–180.
Ashton N. Retinal vascularization in health and disease. Am J Ophthalmol. 1957;44:7–17.
Shimizu K, Kobayashi Y, Muraoka K. Midperipheral fundus involvement in diabetic retinopathy. Ophthalmology. 1981;88:601–612. [CrossRef] [PubMed]
Smith LEH, Kopchick JJ, Chen W, et al. Essential role of growth hormone in ischemia-induced retinal neovascularization. Science. 1997;276:1706–1709. [CrossRef] [PubMed]
Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992;359:843–845. [CrossRef] [PubMed]
Plate KH, Breier G, Welch HA, Risau W. Vascular endothelial growth factor is a potential tumor angiogenesis factor in human gliomas in vivo. Nature. 1992;359:845–848. [CrossRef] [PubMed]
Forsythe JA, Jiang B-H, Iyer NV, et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. 1996;16:4604–4613. [PubMed]
Adamis AP, Miller JW, Bernal M-T, et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol. 1994;118:445–450. [CrossRef] [PubMed]
Aiello LP, Avery RL, Arrigg PG, et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med. 1994;331:1480–1487. [CrossRef] [PubMed]
Malecaze F, Clamens S, Simorre–Pinatel V, et al. Detection of vascular endothelial growth factor messenger RNA and vascular endothelial growth factor-like activity in proliferative diabetic retinopathy. Arch Ophthalmol. 1994;112:1476–1482. [CrossRef] [PubMed]
Pe’er J, Shweiki D, Itin A, Hemo I, Gnessin H, Keshet E. Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases. Lab Invest. 1995;72:638–645. [PubMed]
Miller JW, Adamis AP, Shima DT, et al. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am J Pathol. 1994;145:574–584. [PubMed]
Pierce EA, Avery RL, Foley ED, Aiello LP, Smith LEH. Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization. Proc Natl Acad Sci USA. 1995;92:905–909. [CrossRef] [PubMed]
Okamoto N, Tobe T, Hackett SF, et al. Transgenic mice with increased expression of vascular endothelial growth factor in the retina: a new model of intraretinal and subretinal neovascularization. Am J Pathol. 1997;151:281–291. [PubMed]
Tobe T, Okamoto N, Vinores MA, et al. Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors. Invest Ophthalmol Vis Sci. 1998;39:180–188. [PubMed]
Aiello LP, Pierce EA, Foley ED, et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA. 1995;92:10457–10461. [CrossRef] [PubMed]
Robinson GS, Pierce EA, Rook SL, Foley E, Webb R, Smith LES. Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy. Proc Natl Acad Sci USA. 1996;93:4851–4856. [CrossRef] [PubMed]
Adamis AP, Shima DT, Tolentino MJ, et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization. Arch Ophthalmol. 1996;114:66–71. [CrossRef] [PubMed]
Grunwald J, Hariprasad S, DuPont J, et al. Foveolar choroidal blood flow in age-related macular degeneration. Invest Ophthalmol Vis Sci. 1998;39:385–390. [PubMed]
Ross RD, Barofsky JM, et al. Presumed macular choroidal watershed vascular filling, choroidal neovascularization, and systemic vascular disease in patients with age-related macular degeneration. Am J Ophthalmol. 1998;125:71–80. [CrossRef] [PubMed]
Amin R, Pulkin JE, Frank RN. Growth factor localization in choroidal neovascular membranes of age-related macular degeneration. Invest Ophthalmol Vis Sci. 1994;35:3178–3188. [PubMed]
Frank RN, Amin RH, Eliott D, Puklin JE, Abrams GW. Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes. Am J Ophthalmol. 1996;122:393–403. [CrossRef] [PubMed]
Kvanta A, Algvere PV, Berglin L, Seregard S. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci. 1996;37:1929–1934. [PubMed]
Lopez PF, Sippy BD, Lambert HM, Thach AB, Hinton DR. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. Invest Ophthalmol Vis Sci. 1996;37:855–868. [PubMed]
Ogata N, Matsushima M, Takada Y, et al. Expression of basic fibroblast growth factor mRNA in developing choroidal neovascularization. Curr Eye Res. 1996;15:1008–1018. [CrossRef] [PubMed]
Yi X, Ogata N, Komada M, et al. Vascular endothelial growth factor expression in choroidal neovascularization in rats. Lab Invest. 1997;235:313–319.
Seo M-S, Kwak N, Ozaki H, et al. Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor. Am J Pathol. 1999;154:1743–1753. [CrossRef] [PubMed]
Tobe T, Ortega S, Luna L, et al. Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model. Am J Pathol. 1998;153:1641–1646. [CrossRef] [PubMed]
Bold G, Altmann K-H, Bruggen J, et al. New anilino-phthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor driven angiogenesis. J Med Chem. 2000;43:2310–2323. [CrossRef] [PubMed]
Wood JM, Bold G, Stover D, et al. A novel and potent inhibitor of VEGF receptor tyrosine kinases impairs VEGF-induced responses and tumor growth after oral administration. Cancer Res. 2000;60:2178–2189. [PubMed]
Buchdunger E, Zimmermann J, Mett H, et al. Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class. Proc Natl Acad Sci USA. 1995;92:2558–2562. [CrossRef] [PubMed]
Myllarniemi M, Calderon L, Lemstrom K, Buchdunger E, Hayry P. Inhibition of platelet-derived growth factor receptor tyrosine kinase inhibits vascular smooth muscle cell migration and proliferation. FASEB J. 1997;11:1119–1126. [PubMed]
Fotsis T, Pepper M, Adlercreutz H, et al. Genistein, a dietary-derived inhibitor of in vitro angiogenesis. Proc Natl Acad Sci USA. 1993;90:2690–2694. [CrossRef] [PubMed]
Ozaki H, Okamoto N, Ortega S, et al. Basic fibroblast growth factor is neither necessary nor sufficient for the development of retinal neovascularization. Am J Pathol. 1998;153:757–765. [CrossRef] [PubMed]
Vinores SA, Vinores MA, Chiu C, Woerner TM, Campochiaro PA. Double-labeling for keratin and class III beta-tubulin within cultured retinal pigment epithelial cells: comparison of chromogens to yield maximum resolution of two structural proteins within the same cell. J Histotechnol. 1997;20:19–25. [CrossRef]
Francois J, De Leary JJ, Cambie E, Hanssens M, Victoria–Troncoso V. Neovascularization after argon laser photocoagulation of macular lesions. Am J Ophthalmol. 1975;79:206–210. [CrossRef] [PubMed]
Fine SL, Patz A, Orth DH, Klein MS, Finkelstein D, Yassur Y. Subretinal neovascularization developing after prophylactic argon laser photocoagulation of atrophic macular scars. Am J Ophthalmol. 1976;79:352–357.
Galinos SO, Asdourian GK, Woolf MB, Goldberg M, Busse BJ. Choroido-vitreal neovascularization after argon laser photocoagulation. Arch Ophthalmol. 1775;1975:524–530.
Adamis AP, Shima DT, Yeo TK, et al. Synthesis and secretion of vascular permeability factor/vascular endothelial growth factor by human retinal pigment epithelial cells. Biochem Biophys Res Commun. 1993;193:631–638. [CrossRef] [PubMed]
Mousa SA, Lorelli W, Campochiaro PA. Extracellular matrix-integrin binding modulates secretion of angiogenic growth factors by retinal pigmented epithelial cells. J Cell Biochem. 1999;74:135–143. [CrossRef] [PubMed]
Burns MS, Tyler NK. Selective neovascularization of the retinal pigment epithelium in rat photoreceptor degeneration in vivo. Curr Eye Res. 1990;9:1061–1075. [CrossRef] [PubMed]
Yamada H, Yamada E, Hackett SF, Ozaki H, Okamoto N, Campochiaro PA. Hyperoxia causes decreased expression of vascular endothelial growth factor and endothelial cell apoptosis in adult retina. J Cell Physiol. 1999;179:149–156. [CrossRef] [PubMed]
Ozaki H, Seo M-S, Ozaki K, et al. Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization. Am J Pathol. 2000;156:679–707.
Figure 1.
 
Effect of kinase inhibitors with overlapping activity on CNV in a murine model. Mice with laser-induced rupture of Bruch’s membrane were treated orally by gavage for 2 weeks with vehicle alone (A, E) or with vehicle containing 120 micromoles/kg PTK787 (B, F), CGP 57148 (C, G), or genistein (D, H). Retinal sections from two mice with each treatment were histochemically stained with GSA, which selectively stains vascular cells. Sections were either not counterstained, to provide optimal visualization of retinal vessels (A through D), or were counterstained with contrast blue to allow visualization of retinal neurons (E through H). Arrows: Horizontal extent of the CNV. There were large areas of choroidal neovascularization in mice treated with vehicle alone (A, E) or CGP 57148, the inhibitor of PDGF, but not VEGF receptor kinase activity (C, G). In contrast, mice treated with PTK787, an inhibitor of both VEGF and PDGF receptor kinase activity, had almost no choroidal neovascularization (B, F). Mice treated with genistein, a nonspecific kinase inhibitor, had an intermediate amount of choroidal neovascularization.
Figure 1.
 
Effect of kinase inhibitors with overlapping activity on CNV in a murine model. Mice with laser-induced rupture of Bruch’s membrane were treated orally by gavage for 2 weeks with vehicle alone (A, E) or with vehicle containing 120 micromoles/kg PTK787 (B, F), CGP 57148 (C, G), or genistein (D, H). Retinal sections from two mice with each treatment were histochemically stained with GSA, which selectively stains vascular cells. Sections were either not counterstained, to provide optimal visualization of retinal vessels (A through D), or were counterstained with contrast blue to allow visualization of retinal neurons (E through H). Arrows: Horizontal extent of the CNV. There were large areas of choroidal neovascularization in mice treated with vehicle alone (A, E) or CGP 57148, the inhibitor of PDGF, but not VEGF receptor kinase activity (C, G). In contrast, mice treated with PTK787, an inhibitor of both VEGF and PDGF receptor kinase activity, had almost no choroidal neovascularization (B, F). Mice treated with genistein, a nonspecific kinase inhibitor, had an intermediate amount of choroidal neovascularization.
Figure 2.
 
Integrated area of CNV and box plot of the log values of the integrated area of CNV in control mice and mice treated with PTK787, CGP 53716, or genistein. Mice with laser-induced rupture of Bruch’s membrane were treated orally by gavage with vehicle or 120 micromoles/kg of one of the drugs for 2 weeks. Serial sections through the entire extent of each lesion were stained with GSA, and the area of CNV on each section was measured by image analysis. All the area measurements for each lesion were added together to generate the integrated area of CNV. Mean ± SEM is plotted on the left in the bar graph (n for each treatment group: 52 for control; 52, genistein; 56, PTK787; and 52, CGP 53716). *P < 0.001 by repeated-measures ANOVA for difference from control. The log(integrated area of CNV) for each group is shown in a box plot on the right in which the central line is the median, the box contains all values between the 25th and 75th percentiles, and the lines show the highest and lowest value in each group, except for the control and CGP 53716 group in which the highest are shown by circles. Statistical comparisons by repeated measures analysis of variance showed P < 0.001 for differences between control and PTK787, control and genistein, and PTK787 and genistein. P = 0.56 for difference between control and CGP 53716.
Figure 2.
 
Integrated area of CNV and box plot of the log values of the integrated area of CNV in control mice and mice treated with PTK787, CGP 53716, or genistein. Mice with laser-induced rupture of Bruch’s membrane were treated orally by gavage with vehicle or 120 micromoles/kg of one of the drugs for 2 weeks. Serial sections through the entire extent of each lesion were stained with GSA, and the area of CNV on each section was measured by image analysis. All the area measurements for each lesion were added together to generate the integrated area of CNV. Mean ± SEM is plotted on the left in the bar graph (n for each treatment group: 52 for control; 52, genistein; 56, PTK787; and 52, CGP 53716). *P < 0.001 by repeated-measures ANOVA for difference from control. The log(integrated area of CNV) for each group is shown in a box plot on the right in which the central line is the median, the box contains all values between the 25th and 75th percentiles, and the lines show the highest and lowest value in each group, except for the control and CGP 53716 group in which the highest are shown by circles. Statistical comparisons by repeated measures analysis of variance showed P < 0.001 for differences between control and PTK787, control and genistein, and PTK787 and genistein. P = 0.56 for difference between control and CGP 53716.
Figure 3.
 
Log value of retinal vessel area per section in control mice and mice treated for 2 weeks with 120 micromoles/kg of PTK787, CGP 53716, or genistein. Serial sections were cut through entire eyes and stained with GSA. The retinal vessel area was determined by image analysis on every 10th section, and the mean was calculated for each eye. The box plot and symbols are as described for Figure 2 . Statistical comparisons were made by repeated-measures ANOVA, and there were no significant differences between any members of the group.
Figure 3.
 
Log value of retinal vessel area per section in control mice and mice treated for 2 weeks with 120 micromoles/kg of PTK787, CGP 53716, or genistein. Serial sections were cut through entire eyes and stained with GSA. The retinal vessel area was determined by image analysis on every 10th section, and the mean was calculated for each eye. The box plot and symbols are as described for Figure 2 . Statistical comparisons were made by repeated-measures ANOVA, and there were no significant differences between any members of the group.
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