VEGF Is Major Stimulator in Model of Choroidal Neovascularization

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. 

¹  Present address: The Department of Ophthalmology, the Catholic University of Korea School of Medicine, Seoul.

 Supported by Public Health Service Grants EY05951, EY12609, and Core Grant P30EY1765 (in support of the statistical work at the Wilmer Institute) from the National Eye Institute, a Lew R. Wasserman Merit Award (PAC); an unrestricted grant from Research to Prevent Blindness; a grant from CIBA Vision, Inc., a Novartis company; the Rebecca P. Moon, Charles M. Moon, Jr., and Dr. P. Thomas Manchester Research Fund; a grant from Mrs. Harry J. Duffey; a grant from Dr. and Mrs. William Lake; a grant from Project Insight; and a grant from the Association for Retinopathy of Prematurity and Related Diseases. PAC is the George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience.

 Submitted for publication October 1, 1999; revised December 21, 1999 and February 14, 2000; accepted March 15, 2000.

 Commercial relationships policy: PAC is a consultant for CIBA Vision. The terms of this arrangement are managed by The Johns Hopkins University in accordance with conflict of interest policies C1, C5, C8, N(NK, NO, JMW).

 

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 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.