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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 Ltd.
Pharmaceuticals, Inc., Basel, Switzerland
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Choroidal neovascularization occurs in several diseases in which there are abnormalities of Bruch's membrane. The most prevalent disease of this type is age-related macular degeneration, the most common cause of severe vision loss in patients over the age of 60 in developed countries.3 Neovascularization originating from choroidal vessels grows through Bruch's membrane into the sub-retinal pigmented epithelial space and sometimes into the subretinal space. The blood vessels leak fluid, which collects beneath the retina causing reversible visual loss, and they bleed and cause scarring that results in permanent loss of central vision. Current treatments are designed to destroy or remove the abnormal blood vessels and do not address the underlying stimuli responsible for neovascularization; therefore, recurrent neovascularization and permanent visual loss occur in the majority of patients who initially have successful treatment.3 Drug treatment that blocks the stimuli for choroidal neovascularization would be a major advance, but its development is hindered by our poor understanding of pathogenesis.
More is known about the cascade of events leading to retinal neovascularization than that for choroidal neovascularization, because some of the molecular signals involved in the development of retinal neovascularization have been defined. For instance, several lines of evidence suggest that vascular endothelial growth factor (VEGF) plays an important role in retinal vascularization during development and in pathological neovascularization in ischemic retinopathies. The expression of VEGF is increased by hypoxia,4,5 which is a prominent feature of both of these processes. Stimulated by VEGF released by the avascular, hypoxic peripheral retina, blood vessels begin to develop at the optic nerve and extend to the periphery of the retina.6 Likewise, VEGF participates in pathological retinal neovascularization, because its levels are increased in the retina and vitreous of patients7-10 or laboratory animals11,12 with ischemic retinopathies, and increased expression of VEGF in retinal photoreceptors of transgenic mice stimulates neovascularization within the retina.13 The implication of VEGF in retinal neovascularization led to studies investigating VEGF antagonists in models of ischemic retinopathy. Soluble VEGF receptor/IgG fusion proteins or VEGF antisense oligonucleotides each inhibited retinal neovascularization by ~50% in the murine model of oxygen-induced ischemic retinopathy.14,15 Antibodies to VEGF partially inhibited iris neovascularization in a monkey model of ischemic retinopathy.16
Although VEGF plays a central role, it is not the only stimulator involved, which might explain why VEGF antagonists are only partially effective. Growth hormone acting through insulin-like growth factor (IGF)-I also participates in retinal neovascularization, and decreased IGF-I in genetically engineered mice or antagonism of IGF-I by somatostatin analogs results in approximately a 30% decrease in retinal neovascularization in mice with ischemic retinopathy.17
Intracellular signaling induced by VEGF is complex, but it has been suggested that protein kinase C (PKC), particularly the PKCßII isoform, plays a prominent role.18,19 A specific antagonist of PKCß isoforms partially inhibits retinal neovascularization after laser-induced branch vein occlusion.20
Integrins 
vß3 and
vß5 are induced on endothelial cells,
including those in the retina, participating in
neovascularization.21,22
Two independent studies using
different peptides that antagonize binding to
vß3 or both
vß3 and vß5
each demonstrated up to 50% inhibition of retinal neovascularization
in the murine model of oxygen-induced ischemic
retinopathy.22,23
Thus, several different types of agents that work by different mechanisms can cause partial inhibition of retinal neovascularization. This suggests that drug treatment of retinal neovascularization in patients may be feasible, but there are several questions related to this issue that remain. For instance, why is it that 50% inhibition has been the maximal achievable limit with several types of agents given by different routes of administration, including intraocular injections? Is there so much redundancy built into the retinal neovascularization cascade that this is the most that can be attained, and if so, is it sufficient to provide clinical benefit? Would an agent or combination of agents that act on multiple targets in the cascade be more effective? Do any of the molecular signals implicated in retinal neovascularization also play a role in choroidal neovascularization, and are there agents that inhibit both?
PKC consists of a family of at least 10 related serine/threonine kinases.24 Staurosporine is an alkaloid produced by bacteria that is a potent nonspecific inhibitor of PKC25 that also inhibits other serine/threonine kinases, such as protein kinase A (PKA), and tyrosine kinases, such as epidermal growth factor receptor (EGFR).26 CGP 41251 is a derivative of staurosporine with the chemical name N-benzoyl-staurosporine that was developed as a PKC inhibitor for treatment of cancer.26 It is a less potent inhibitor of PKC than staurosporine but is more specific because it is a weak inhibitor of PKA and EGFR, and it has been used in several studies to assess the role of PKC in cellular functions.27-30 Recently, one of the authors (J.M. Wood) determined that CGP 41251 is also a relatively potent inhibitor of VEGF and platelet-derived growth factor (PDGF) receptor tyrosine kinases (unpublished data). It was also shown to inhibit VEGF-induced angiogenesis in a mouse subcutaneous growth factor implant model. As antagonism of VEGF and inhibition of PKC, each have been demonstrated to partially inhibit retinal neovascularization14,15,20 and CGP 41251 has both activities, we investigated the effect of CGP 41251 in animal models of retinal and choroidal neovascularization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of CGP 41251 on the enzymatic activity of several members of the PKC family was measured using purified enzymes and artificial substrates as previously described.29 The effect of CGP 41251 on phosphorylation of VEGFRs and other tyrosine kinase receptors was measured using purified recombinant glutathione S-transferase (GST)-fused kinase domains in the presence of substrate and labeled ATP.31 The kinase domain-fusion proteins were expressed in baculovirus, purified over glutathione-Sepharose, and diluted in 10 mmol/L Tris/HCl (pH 7.2) based on their specific activity to obtain an activity of 4000 to 6000 cpm above background (<400 cpm). [33P]ATP (Amersham, Arlington Heights, IL) was used as the phosphate donor, and the polyGluTyr(4:1) peptide (P-275, Sigma Chemical Co., St. Louis, MO) was used as the acceptor. CGP 41251 was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mmol/L and then diluted as required so that the final DMSO concentration was 1%. The assay mixture, which was optimized for each kinase (20 mmol/L Tris/HCl (pH 7.5), 1 to 10 mmol/L MnCl2, 1 to 10 mmol/L MgCl2, 1 to 8 µmol/L ATP, 0.2 µCi of [33P]ATP, 3 to 8 µg/ml polyGluTyr(4:1)) was incubated with the respective GST-fused kinase with or without CGP 41251 for 10 minutes at room temperature in a total volume of 30 µl. The reaction was stopped by adding 10 µl of 250 mmol/L EDTA. Using a 96-well filter system (GIBCO BRL, Gaithersburg, MD), 20 µl of the reaction mixture was transferred onto an Immobilon-PVDF membrane (IPVH 000 10, Millipore, Bedford, MA). Membranes were washed extensively with 0.5% H3PO4 and soaked in ethanol. After drying, Microscint cocktail (TM-0 6013611, Packard, Meriden, CT) was added, and scintillation counting was performed (Hewlett Packard Top Count). IC50 values were calculated by linear regression analysis of the percentage inhibition by CGP 41251 over a range of different concentrations
Drug Treatment of Mice with Ischemic Retinopathy
Ischemic retinopathy was produced in C57BL/6J mice by a method described by Smith et al.32 Seven-day-old (P7) mice and their mothers were placed in an airtight incubator and exposed to an atmosphere of 75 ± 3% oxygen for 5 days. Incubator temperature was maintained at 23 ± 2°C, and oxygen was measured every 8 hours with an oxygen analyzer. After 5 days, the mice were removed from the incubator and placed in room air, and drug treatment was begun. Drug was dissolved in DMSO and diluted to final concentrations with water; the maximal concentration of DMSO was 1%. Vehicle (1% DMSO) or vehicle containing various concentrations of drug (volume = 10 µl per gram body weight) was placed in the stomach by gavage once a day. At P17, after 5 days of treatment, mice were sacrificed, and eyes were rapidly removed and frozen in optimal cutting temperature embedding compound (OCT; Miles Diagnostics, Elkhart, IN) or fixed in 10% phosphate-buffered formalin and embedded in paraffin. Adult C57BL/6J mice were also treated by gavage with drug or vehicle, and after 5 days, they were sacrificed and their eyes were processed for frozen or paraffin sections.
Quantitation of Retinal Neovascularization
Frozen sections (10 µm) of eyes from drug-treated and control mice were histochemically stained with biotinylated griffonia simplicifolia lectin B4 (GSA, Vector Laboratories, Burlingame, CA), which selectively binds to endothelial cells. Slides were incubated in methanol/H2O2 for 10 minutes at 4°C, washed with 0.05 mol/L Tris-buffered saline, pH 7.6 (TBS), and incubated for 30 minutes in 10% normal porcine serum. Slides were incubated for 2 hours at room temperature with biotinylated GSA, and after rinsing with 0.05 mol/L TBS, they were incubated with avidin coupled to peroxidase (Vector Laboratories) for 45 minutes at room temperature. After being washed for 10 minutes with 0.05 mol/L TBS, slides were incubated with diaminobenzidine to give a brown reaction product. Some slides were counterstained with hematoxylin, and all were mounted with Cytoseal.
To perform quantitative assessments, 10-µm serial sections were cut through one-half of each eye, and sections roughly 50 to 60 µm apart were stained with GSA, providing 13 sections per eye for analysis. GSA-stained sections were examined with an Axioskop microscope (Zeiss, Thornwood, NY), and images were digitized using a 3 CCD color video camera (IK-TU40A, Toshiba, Tokyo, Japan) and a frame grabber. Image-Pro Plus software (Media Cybernetics, Silver Springs, MD) was used to delineate GSA-stained cells on the surface of the retina, and their area was measured. The mean of the 13 measurements from each eye was used as a single experimental value.
Drug Treatment of Mice during Retinal Vascular Development
Litters of newborn C57BL/6J mice were divided into treatment and control groups that received daily subcutaneous injections of 100 mg/kg drug or vehicle, respectively. At P7 or P10, mice were anesthetized and perfused with 1 ml of phosphate-buffered saline containing 50 mg/ml fluorescein-labeled dextran (2 x 106 average molecular weight; Sigma) as previously described.33 The eyes were removed and fixed for 1 hour in 10% phosphate-buffered formalin. The cornea and lens were removed and the entire retina was carefully dissected from the eyecup. Radial cuts were made from the edge of the retina to the equator in all four quadrants, and the retina was flat mounted in Aquamount with photoreceptors facing upward. Flat mounts were examined by fluorescence microscopy, and images were digitized using a 3 CCD color video camera and a frame grabber. Image-Pro Plus was used to measure the distance from the center of the optic nerve to the leading front of developing retinal vessels in each quadrant, and the mean was used as a single experimental value.
Drug Treatment of Mice with Laser-Induced Choroidal Neovascularization
Choroidal neovascularization was generated by modification of a previously described technique.34 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. Three burns of krypton laser photocoagulation (100-µm spot size, 0.1-second duration, 150 mW) were delivered to each retina using the slit lamp delivery system of a Coherent model 920 photocoagulator 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 choroidal neovascularization,34 so only mice in which a bubble was produced for all three burns were included in the study. Ten mice were randomly assigned to treatment with vehicle alone, and 10 mice received vehicle containing 400 mg/kg/day of CGP 41251 orally by gavage. After 14 days, the mice were killed with an overdose of pentobarbital sodium, and their eyes were rapidly removed and frozen in OCT.
Quantitative Analysis of the Amount of Choroidal Neovascularization
Frozen serial sections (10 µm) were cut through the entire extent of each burn and histochemically stained with biotinylated GSA as described above. Histomark Red (Kirkegaard and Perry, Gaithersburg, MD) was used as chromogen to give a red reaction product that is distinguishable from melanin. Some slides were counterstained with Contrast Blue (Kirkegaard and Perry).
To perform quantitative assessments, GSA-stained sections were examined with an Axioskop microscope, and images were digitized using a 3 CCD color video camera and a frame grabber. Image-Pro Plus software was used to delineate and measure the area of GSA-stained blood vessels in the subretinal space. For each 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 lesions in which good sections were obtained through the entire lesion, so that a valid area measurement could be made on each, were included in the analysis. There appeared to be little variability among lesions in individual mice, and all excluded lesions were qualitatively similar in size to included lesions and were excluded solely due to inability to obtain an accurate measurement because of poor quality of some sections. Values were averaged to give one experimental value per mouse. A two-sample t-test for unequal variances was performed to compare the log mean integrated area between treated and control mice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Table 1
shows the kinase inhibitory
profile of CGP 41251. The IC50 for several subtypes of PKC as well as
the KDR tyrosine kinase of human VEGF receptor-2 and the tyrosine
kinase of human PDGF receptor-ß are in the same range (20 to 100
nmol/L). At approximately 10-fold higher concentrations, CGP 41251
inhibits Flk-1, the tyrosine kinase of the mouse VEGF receptor
corresponding to human KDR, and Flt-1, the tyrosine kinase of human
VEGF receptor-1. The IC50s for other receptor tyrosine kinases, such as
Tie 2, fibroblast growth factor receptor-1, or epidermal growth factor
receptor, are 3 µmol/L or above.
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Retinas of nonischemic P17 mice stained with GSA show normal
vessels in the superficial and deep capillary beds with a few
connecting vessels (Figure 1, A and B)
.
P17 mice with ischemic retinopathy treated with vehicle show a marked
increase in the area of endothelial cell staining throughout the retina
with large clumps of cells on the retinal surface (Figure 1, C and D)
,
not seen in nonischemic retinas. P17 mice with ischemic retinopathy
given 600 mg/kg CGP 41251 once a day for 5 days by gavage have a
dramatic decrease in endothelial cell staining on the surface and
within the retina (Figure 1, E and F)
compared with vehicle-treated
mice. In fact, the endothelial cell staining within the retina is less
than that seen in nonischemic P17 mice. High magnification shows that
there are no identifiable endothelial cells on the surface of the
retina, indicating that there is complete inhibition of
neovascularization (Figure 1F)
. There is also a striking absence of
endothelial cell staining in the inner nuclear layer and outer
plexiform layer where the deep capillary beds are normally located. P17
mice with ischemic retinopathy given 300 mg/kg (Figure 1G)
or 60 mg/kg
(Figure 1H)
CGP 41251 once a day by gavage show some clumps of
neovascularization on the surface of the retina (arrows) that are less
than those seen in vehicle-treated controls. They also show some
decrease in endothelial staining within the retina, but there is some
present (arrowheads). Image analysis demonstrates that mice treated
once a day with 600, 60, or 6 mg/kg CGP 41251 show a statistically
significant dose-dependent decrease in endothelial cell staining on and
in the retinas compared with vehicle-treated mice
(P < 0.001 by analysis of variance (ANOVA);
Figure 2
).
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The decrease in endothelial staining in the inner nuclear layer of
the retinas of CGP-41251-treated mice suggests that there might be some
inhibition of retinal vascular development, because the time of
treatment (P12 to P17) corresponds to the period of development of the
deep capillary bed.35
To test this, neonatal mice were
treated with subcutaneous injections of 100 mg/kg CGP 41251 or vehicle
alone starting on P0, and on P7 or P10 they were perfused with
fluorescein-labeled dextran and retinal whole mounts were prepared. At
P7, retinal vessels in vehicle-treated mice have almost reached the
peripheral edge of retina (Figure 3, A and C)
, but in CGP-41251-treated mice, the retinal vessels have
extended only slightly more than halfway to the periphery (Figure 3, B and D)
. At P10, in vehicle-treated mice, the superficial capillary bed
is complete and extends all of the way to the peripheral edge of the
retina, and the deep capillary bed is partially developed. But in
CGP-41251-treated mice, the superficial capillary bed has not yet
reached the edge of the retina (not shown). The distance from the optic
nerve to the vascular front was calculated by image analysis, and the
differences between treated and control mice at P7 and P10 were
statistically significant (Figure 4)
.
This indicates that CGP 41251 inhibits normal retinal vascular
development as well as pathological retinal neovascularization.
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Adult mice were given 600 mg/kg CGP 41251 by gavage once a day for
5 days, the highest dose used in the model of oxygen-induced ischemic
retinopathy. There was no difference in the total area of endothelial
staining in the retina or the appearance of retinal vessels in
CGP-41251-treated mice compared with vehicle-treated mice (Figure 5, A and B)
. Image analysis shows no
difference in the amount of retinal endothelial cell staining between
CGP-41251- and vehicle-treated mice. This suggests that CGP 41251 is
not toxic to endothelial cells of mature vessels.
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Two weeks after laser photocoagulation, all lesions in both groups
of mice showed a discontinuity in Bruch's membrane with roughly
equivalent damage to the overlying retina. All mice treated with
vehicle alone showed large areas of choroidal neovascularization at the
site of each laser-induced rupture of Bruch's membrane (Figure 6, A and C)
. There was proliferation of
retinal pigmented epithelial cells along the margin of the new vessels.
Retinal blood vessels stained with GSA were seen in the overlying
retina. In contrast, all mice given 400 mg/kg/day CGP 41251 by gavage
had very little if any choroidal neovascularization at the site of each
laser-induced rupture of Bruch's membrane. In many instances, there
was no identifiable GSA-stained neovascular tissue throughout the
entire burn (Figure 6D)
, but some burns contained regions in which
there were thin disks of GSA-stained tissue (Figure 6B)
. There was mild
proliferation of retinal pigmented epithelial cells. Despite the marked
decrease in choroidal neovascularization in the eyes of treated mice,
the overlying retinal vessels appeared normal. This was best seen
in sections with no counterstain (Figure 6B)
.
|
shows the measurements for each of the
lesions in which intact, adequately stained sections were obtained all
of the way through the lesion, allowing measurement of the area of
neovascularization on each section. In all but four mice, high quality,
well stained inclusive sections were obtained for at least four of six
lesions (two mice in the treated group and one in the control group had
three measurable lesions, and one control mouse had two measurable
lesions). Qualitatively, all of the lesions in an individual were very
similar in size, with no systematic differences between included and
excluded lesions except for the inability to obtain accurate
quantitative assessment for excluded lesions. The mean of the
measurements made in each individual mouse constituted a single
experimental value. The mean integrated area of choroidal
neovascularization in 10 CGP 41251 mice was 0.0090182 ± 0.0017540
mm2
compared with 0.0695621 ± 0.0073960
mm2
in 10 control mice, nearly an eightfold difference.
This difference was highly statistically significant
(P = 0.004; Figure 7
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that several pharmacological agents
with different mechanisms of action are capable of partially inhibiting
retinal neovascularization. Antagonism of VEGF by soluble VEGF
receptors coupled to IgG heavy chains14
or by antisense
oligonucleotides15
or blockade of integrin
vß3 by two different cyclic
peptides22,23
each inhibit retinal neovascularization in
the murine model of oxygen-induced ischemic retinopathy by as much as
50%. In this study, using the same model, we have demonstrated for the
first time that it is possible to achieve essentially complete
inhibition of retinal neovascularization by drug treatment. This
provides strong support for the feasibility of using drugs to treat
retinal neovascularization in patients with proliferative diabetic
retinopathy or other blinding ischemic retinopathies. It is
particularly encouraging that this dramatic treatment effect was
achieved with oral administration, the preferred route of
administration in patients.
The drug used in this study, CGP 41251, is an inhibitor of several PKC isoforms, including PKCßII, which has been implicated in diabetic complications, including neovascularization in the retina.19,20 CGP 41251 is also an inhibitor of phosphorylation by VEGF and PDGF receptors. As previous studies with agents that specifically block PKCß isoforms or specifically antagonize VEGF have resulted in only partial inhibition of retinal neovascularization, it may be that the greater efficacy of CGP 41251 is due to an additive effect of these different activities. However, it is also possible that the inhibitory effect on retinal neovascularization occurs predominantly through one of these activities and the greater efficacy of CGP 41251 compared with the more specific agents is due to a difference in pharmacokinetics or a difference in mechanism of action. For instance, it could be that blockade of VEGF receptor phosphorylation is a more effective way to block VEGF signaling than attempting to limit the amount of VEGF available to bind to the receptor.14,15 In any case, this study demonstrates that it is possible to achieve much better inhibition of retinal neovascularization by pharmacological means than was previously demonstrated, and additional work is needed to determine the mechanism through which this occurs so that additional effective drugs can be designed. Identification and testing of drugs with different kinase inhibitory activities may help to accomplish this goal.
Our data also indicate that CGP 41251 inhibits normal retinal vascular development in addition to pathological retinal neovascularization. Although we were surprised by this finding initially, because it was not reported to occur from treatment with other VEGF antagonists in the same model,14,15 its occurrence is understandable, because VEGF has been demonstrated to be a critical stimulator of normal retinal vascular development.6 Down-regulation of VEGF during development by hyperoxia arrests vascular development and causes vaso-occlusion.37 When hyperoxia-induced blockade is followed by pharmacological blockade of VEGF signaling (as well as PDGF and PKC signaling), it is not surprising that parts of the retina never develop retinal vessels.
More is known regarding the molecular signals involved in retinal neovascularization than those involved in choroidal neovascularization. This and lack of an inexpensive animal model in which it is possible to precisely measure the amount of choroidal neovascularization have hindered identification of agents that inhibit choroidal neovascularization. We have recently adapted to mice34 a model of laser-induced choroidal neovascularization that was first developed in primates.38 In this study, we report a technique of quantitatively assessing the amount of choroidal neovascularization, providing a means to objectively assess drug effects. Using this approach, we showed that oral administration of 400 mg/kg CGP 41251 dramatically inhibits choroidal neovascularization. This suggests that activation of PKC and/or VEGF signaling and/or PDGF signaling are involved in development of choroidal neovascularization in this model. By using drugs with different, but overlapping in vitro activities, it should be possible to further define the molecular signals involved in choroidal neovascularization.
The results of this study are encouraging in two other respects. CGP 41251 is the first drug identified to have a strong inhibitory effect on both retinal and choroidal neovascularization. Perhaps inhibitory activity in models of retinal neovascularization will have some predictive value for treatment of choroidal neovascularization. Additional correlative studies are needed to determine whether this is actually the case, but if so, it will simplify screening of drugs for ocular neovascularization. Second, although CGP 41251 is the first kinase inhibitor to be evaluated for its effect on retinal and choroidal neovascularization, and other agents in this class of drugs need to be evaluated and could possibly be more potent, the effects with CGP 41251 are dramatic and suggest that it may be useful for treatment of patients with retinal or choroidal neovascularization. The inhibitory effect of CGP 41521 on normal retinal vascular development may preclude its use in infants with retinopathy of prematurity, but the results of our study predict that CGP 41251 is a good candidate for treatment of adults with proliferative diabetic retinopathy and other ischemic retinopathies or choroidal neovascularization due to macular degeneration, ocular histoplasmosis, or a host of other diseases. Toxicity studies in animals have not identified any adverse systemic effects of orally administered CGP 41251 (unpublished data), and phase I clinical trials are underway in cancer patients. This study suggests that clinical trials in patients with retinal or choroidal neovascularization should also be considered.
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Acknowledgements |
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Footnotes |
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Supported by U.S. Public Health Service grants EY05951 and EY2609 and core grant P30EY1765 from the National Eye Institute, a Lew R. Wasserman Merit Award (P.A. Campochiaro) and an unrestricted grant from Research to Prevent Blindness, Inc., a Juvenile Diabetes Foundation fellowship grant (N. Okamoto), 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. P.A. Campochiaro is the George S. and Dolores D. Eccles Professor of Ophthalmology and Neuroscience.
CIBA Vision provided partial funding of the study reported in this article. P.A. Campochiaro is a consultant to CIBA Vision. The terms of this arrangement are being managed by The Johns Hopkins University in accordance with the conflict of interest policies.
M.S. Seo and N. Kwak contributed equally to the manuscript.
M.S. Seo's current address: Chonnam National University Medical School and Hospital, Kwangju, Korea.
N. Kwak's current address: Department of Ophthalmology, the Catholic University of Korea School of Medicine.
H. Ozaki's current address: Fukuoka University School of Medicine, Fukuoka, Japan.
Accepted for publication February 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-v ß-3 for angiogenesis. Science 1994, 264:569-571-v ß-3 inhibit retinal neovascularization in a murine model. Lab Invest 1996, 75:563-573[Medline]
in the induction of ß-casein expression. Cell Growth Differ 1994, 5:239-247[Abstract]
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Y. Saishin, R. L. Silva, Y. Saishin, K. Callahan, C. Schoch, M. Ahlheim, H. Lai, F. Kane, R. K. Brazzell, D. Bodmer, et al. Periocular Injection of Microspheres Containing PKC412 Inhibits Choroidal Neovascularization in a Porcine Model Invest. Ophthalmol. Vis. Sci., November 1, 2003; 44(11): 4989 - 4993. [Abstract] [Full Text] [PDF]
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T. A. Ciulla, A. G. Amador, and B. Zinman Diabetic Retinopathy and Diabetic Macular Edema: Pathophysiology, screening, and novel therapies Diabetes Care, September 1, 2003; 26(9): 2653 - 2664. [Abstract] [Full Text] [PDF]
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H. Nambu, R. Nambu, M. Melia, and P. A. Campochiaro Combretastatin A-4 Phosphate Suppresses Development and Induces Regression of Choroidal Neovascularization Invest. Ophthalmol. Vis. Sci., August 1, 2003; 44(8): 3650 - 3655. [Abstract] [Full Text] [PDF]
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Y. Saishin, Y. Saishin, K. Takahashi, M.-S. Seo, M. Melia, and P. A. Campochiaro The Kinase Inhibitor PKC412 Suppresses Epiretinal Membrane Formation and Retinal Detachment in Mice with Proliferative Retinopathies Invest. Ophthalmol. Vis. Sci., August 1, 2003; 44(8): 3656 - 3662. [Abstract] [Full Text] [PDF]
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P. G. McGuire, T. R. Jones, N. Talarico, E. Warren, and A. Das The Urokinase/Urokinase Receptor System in Retinal Neovascularization: Inhibition by A6 Suggests a New Therapeutic Target Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2736 - 2742. [Abstract] [Full Text] [PDF]
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F. Wang, K. G. Rendahl, W. C. Manning, D. Quiroz, M. Coyne, and S. S. Miller AAV-Mediated Expression of Vascular Endothelial Growth Factor Induces Choroidal Neovascularization in Rat Invest. Ophthalmol. Vis. Sci., February 1, 2003; 44(2): 781 - 790. [Abstract] [Full Text] [PDF]
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K. Takahashi, Y. Saishin, Y. Saishin, K. Mori, A. Ando, S. Yamamoto, Y. Oshima, H. Nambu, M. B. Melia, D. P. Bingaman, et al. Topical Nepafenac Inhibits Ocular Neovascularization Invest. Ophthalmol. Vis. Sci., January 1, 2003; 44(1): 409 - 415. [Abstract] [Full Text] [PDF]
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 E S Bamberger and C W Perrett Angiogenesis in epithelian ovarian cancer Mol. Pathol., December 1, 2002; 55(6): 348 - 359. [Abstract] [Full Text] [PDF]
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A. Ando, A. Yang, H. Nambu, and P. A. Campochiaro Blockade of Nitric-Oxide Synthase Reduces Choroidal Neovascularization Mol. Pharmacol., September 1, 2002; 62(3): 539 - 544. [Abstract] [Full Text] [PDF]
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K. Mori, P. Gehlbach, A. Ando, D. McVey, L. Wei, and P. A. Campochiaro Regression of Ocular Neovascularization in Response to Increased Expression of Pigment Epithelium-Derived Factor Invest. Ophthalmol. Vis. Sci., July 1, 2002; 43(7): 2428 - 2434. [Abstract] [Full Text] [PDF]
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K. Mori, P. Gehlbach, S. Yamamoto, E. Duh, D. J. Zack, Q. Li, K. I. Berns, B. J. Raisler, W. W. Hauswirth, and P. A. Campochiaro AAV-Mediated Gene Transfer of Pigment Epithelium-Derived Factor Inhibits Choroidal Neovascularization Invest. Ophthalmol. Vis. Sci., June 1, 2002; 43(6): 1994 - 2000. [Abstract] [Full Text] [PDF]
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K. Mori, P. Gehlbach, A. Ando, G. Dyer, E. Lipinsky, A. G. Chaudhry, S. F. Hackett, and P. A. Campochiaro Retina-Specific Expression of PDGF-B Versus PDGF-A: Vascular Versus Nonvascular Proliferative Retinopathy Invest. Ophthalmol. Vis. Sci., June 1, 2002; 43(6): 2001 - 2006. [Abstract] [Full Text] [PDF]
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E. J. Duh, H. S. Yang, I. Suzuma, M. Miyagi, E. Youngman, K. Mori, M. Katai, L. Yan, K. Suzuma, K. West, et al. Pigment Epithelium-Derived Factor Suppresses Ischemia-Induced Retinal Neovascularization and VEGF-Induced Migration and Growth Invest. Ophthalmol. Vis. Sci., March 1, 2002; 43(3): 821 - 829. [Abstract] [Full Text] [PDF]
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 J. Griggs, J. N. Skepper, G. A. Smith, K. M. Brindle, J. C. Metcalfe, and R. Hesketh Inhibition of Proliferative Retinopathy by the Anti-Vascular Agent Combretastatin-A4 Am. J. Pathol., March 1, 2002; 160(3): 1097 - 1103. [Abstract] [Full Text] [PDF]
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D. S. McLeod, M. Taomoto, J. Cao, Z. Zhu, L. Witte, and G. A. Lutty Localization of VEGF Receptor-2 (KDR/Flk-1) and Effects of Blocking It in Oxygen-Induced Retinopathy Invest. Ophthalmol. Vis. Sci., February 1, 2002; 43(2): 474 - 482. [Abstract] [Full Text] [PDF]
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K. Ohno-Matsui, A. Hirose, S. Yamamoto, J. Saikia, N. Okamoto, P. Gehlbach, E. J. Duh, S. Hackett, M. Chang, D. Bok, et al. Inducible Expression of Vascular Endothelial Growth Factor in Adult Mice Causes Severe Proliferative Retinopathy and Retinal Detachment Am. J. Pathol., February 1, 2002; 160(2): 711 - 719. [Abstract] [Full Text] [PDF]
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 J.N. UPALAKALIN, I. HEMO, C. DEHIO, E. KESHET, and L.E. BENJAMIN Survival Mechanisms of VEGF and PlGF during Microvascular Remodeling Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 181 - 188. [Abstract] [PDF]
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 I. Chowers, E. Banin, Y. Hemo, R. Porat, H. Falk, E. Keshet, J. Pe'er, and A. Panet Gene transfer by viral vectors into blood vessels in a rat model of retinopathy of prematurity Br. J. Ophthalmol., August 1, 2001; 85(8): 991 - 995. [Abstract] [Full Text] [PDF]
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K. Mori, A. Ando, P. Gehlbach, D. Nesbitt, K. Takahashi, D. Goldsteen, M. Penn, C. T. Chen, K. Mori, M. Melia, et al. Inhibition of Choroidal Neovascularization by Intravenous Injection of Adenoviral Vectors Expressing Secretable Endostatin Am. J. Pathol., July 1, 2001; 159(1): 313 - 320. [Abstract] [Full Text] [PDF]
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 D. J. Propper, A. C. McDonald, A. Man, P. Thavasu, F. Balkwill, J. P. Braybrooke, F. Caponigro, P. Graf, C. Dutreix, R. Blackie, et al. Phase I and Pharmacokinetic Study of PKC412, an Inhibitor of Protein Kinase C J. Clin. Oncol., March 1, 2001; 19(5): 1485 - 1492. [Abstract] [Full Text] [PDF]
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M. Lonchampt, L. Pennel, and J. Duhault Hyperoxia/Normoxia-Driven Retinal Angiogenesis in Mice: A Role for Angiotensin II Invest. Ophthalmol. Vis. Sci., February 1, 2001; 42(2): 429 - 432. [Abstract] [Full Text]
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V. Stellmach, S. E. Crawford, W. Zhou, and N. Bouck Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor PNAS, January 23, 2001; (2001) 31252398. [Abstract] [Full Text]
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N. Kwak, N. Okamoto, J. M. Wood, and P. A. Campochiaro VEGF Is Major Stimulator in Model of Choroidal Neovascularization Invest. Ophthalmol. Vis. Sci., September 1, 2000; 41(10): 3158 - 3164. [Abstract] [Full Text]
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 P. Parsons-Wingerter, K. E. Elliott, J. I. Clark, and A. G. Farr Fibroblast Growth Factor-2 Selectively Stimulates Angiogenesis of Small Vessels in Arterial Tree Arterioscler. Thromb. Vasc. Biol., May 1, 2000; 20(5): 1250 - 1256. [Abstract] [Full Text] [PDF]
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 R. S. Kerbel Tumor angiogenesis: past, present and the near future Carcinogenesis, March 1, 2000; 21(3): 505 - 515. [Abstract] [Full Text] [PDF]
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H. Ozaki, M.-S. Seo, K. Ozaki, H. Yamada, E. Yamada, N. Okamoto, F. Hofmann, J. M. Wood, and P. A. Campochiaro Blockade of Vascular Endothelial Cell Growth Factor Receptor Signaling Is Sufficient to Completely Prevent Retinal Neovascularization Am. J. Pathol., February 1, 2000; 156(2): 697 - 707. [Abstract] [Full Text] [PDF]
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V. Stellmach, S. E. Crawford, W. Zhou, and N. Bouck Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor PNAS, February 27, 2001; 98(5): 2593 - 2597. [Abstract] [Full Text] [PDF]
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