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Regular Articles |
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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Abstract |
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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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The retina is a tissue with very high metabolic activity that is oxygenated from retinal and choroidal circulations, which each originate from branches of the ophthalmic artery. The choroidal circulation is derived from the long and short posterior ciliary arteries, which pierce the sclera and form successively smaller branches that supply the choriocapillaris, fenestrated microvessels separated from the retina by the retinal pigmented epithelium (RPE). The photoreceptor layer of the retina has no blood vessels and receives oxygen by diffusion from the choriocapillaris. The retinal circulation is derived from the central retinal artery, which enters the eye through the optic nerve and branches to form retinal arterioles that run along the surface of the retina and give rise to the superficial capillary bed. The arterioles also send penetrating branches throughout the inner two-thirds of the retina, which form the intermediate and deep retinal capillary beds.
The retinal circulation develops first at the optic nerve and extends to the periphery along the surface of the retina by vasculogenesis, the formation of blood vessels from pre-existent precursor cells. Blood vessels sprout from the superficial retinal vessels and invade the retina by a process referred to as angiogenesis, resulting in formation of the intermediate and deep capillary beds. Therefore, retinal vascular development involves both vasculogenesis and angiogenesis and occurs late, compared to most other developmental processes. It is completed shortly before term in humans; in several species, including rats and mice, it is completed after birth. Hypoxia in the avascular peripheral retina results in up-regulation of vascular endothelial growth factor (VEGF).1 Hyperoxia inhibits development of retinal blood vessels, and in fact causes them to regress due to apoptosis of vascular endothelial cells.2 This regression is accompanied by down-regulation of VEGF and is prevented by administration of exogenous VEGF. These data suggest that VEGF plays an important role in retinal vascular development.
Neonatal animals with hyperoxia-induced regression of retinal vessels, when removed from hyperoxia and put back into room air, develop severe retinal hypoxia, dramatic up-regulation of VEGF, and retinal NV.3,4 This situation models that of retinopathy of prematurity (ROP) in humans, but also shares features with several disease processes in adults in which retinal vessels become damaged and occluded, leading to retinal ischemia. These diseases are collectively referred to as ischemic retinopathies and include branch retinal vein occlusion, central retinal vein occlusion, and proliferative diabetic retinopathy, the most common cause of severe visual loss in people under 60 in developed countries.5 Hypoxia-induced up-regulation of VEGF has also been implicated in the development of retinal NV in these diseases.6-11
These data suggest that interruption of VEGF signaling is a good target for pharmacological treatment of retinal NV. This has been borne out by studies in which VEGF antagonists have been injected into the eyes of animals with ischemic retinopathies and have caused partial inhibition of retinal NV.12,13 Although these studies confirm that VEGF plays a central role, questions remain as to why VEGF antagonists are only partially effective. It may be that the antagonists are not sufficiently potent or effective levels are short-lived. Delivery is an issue because intraocular injections in mouse eyes are technically difficult and potentially unreliable. However, insulin-like growth factor-I (IGF-I) also contributes to retinal NV in ischemic retinopathies;14 IGF-I, and possibly other growth factors, could be responsible for residual angiogenic activity.
Recently, we demonstrated that a partially selective kinase inhibitor that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors and several isoforms of protein kinase C (PKC) completely inhibits retinal NV.15 In this study, we have used drugs that selectively inhibit tyrosine kinase activity of VEGF and/or PDGF receptors to explore the role of these signaling pathways in retinal vascular development and pathological retinal NV.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ischemic retinopathy was produced in C57BL/6J mice by a method described by Smith et al.3 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 aqueous buffer or dimethyl sulfoxide (DMSO) depending on solubility and diluted to final concentrations with water; the maximum concentration of DMSO was 1%. Vehicle (aqueous buffer or 1% DMSO) or vehicle containing various concentrations of drug (volume, 10 µl/gram body weight) was placed in the stomach by gavage twice a day. At P17, after 5 days of treatment, mice were sacrificed and their eyes rapidly removed and frozen in optimum 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, sacrificed after 5 days, and their eyes 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 (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 (TBS), pH 7.6, and incubated for 30 minutes in 10% normal porcine serum. Slides were incubated 2 hours at room temperature with biotinylated lectin and, after rinsing with 0.05 mol/L TBS, 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 (Stephens Scientific, Riverdale, NJ).
To perform quantitative assessments, 10-µm serial sections were cut through half of each eye, and sections roughly 50 to 60 µm apart were stained with lectin, providing 13 sections per eye for analysis. Lectin-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 Spring, MD) was used to delineate lectin-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 which received daily subcutaneous injections of 10 mg/kg of drug or vehicle, respectively. At P7 or P10, mice were anesthetized with ether and perfused with 1 ml of phosphate-buffered saline containing 50 mg/ml of fluorescein-labeled dextran (2 x 106 average molecular weight, Sigma, St. Louis, MO) as previously described.16 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, radially cut from the edge of the retina to the equator in all four quadrants, and flat-mounted in Aquamount with photoreceptors facing downward. Flat mounts were examined by fluorescence microscopy and images were digitized using a 3 CCD color video camera (IK-TU40A, Toshiba) and a frame grabber. Image-Pro Plus software (Media Cybernetics) 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.
Treatment of Rhodopsin/VEGF Transgenic Mice
Rhodopsin/VEGF transgenic mice16,17 were treated with drug or vehicle by gavage for 2 weeks from P7 to P21. The mice were then anesthetized and perfused with fluorescein-labeled dextran as described above. Retinas were flat mounted with the photoreceptor side up and examined by fluorescence microscopy at 200x magnification, which provides a narrow depth of field, so that when focusing on NV along the outer edge of the retina, the remainder of the retinal vessels are out of focus. This permits easy delineation of the NV. The outer edge of the retina, which corresponds to the subretinal space in vivo, is easily identified; therefore, there is standardization of focal plane from slide to slide. Images were digitized using a 3 CCD color video camera and a frame grabber. Image-Pro Plus was used to delineate each of the lesions and calculate the number in each retina, the area of each lesion, and the total area of NV per retina as previously described.16
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inhibitory profiles of the three partially selective kinase
inhibitors used in this study have been published.18-21
PTK787/ZK 222584 (PTK787) is an excellent inhibitor of phosphorylation
by human VEGF receptor 2 and its mouse homologue, Flk-1
(IC50s: 0.10 and 0.27 µmol/L, respectively). It
is a less potent inhibitor of VEGF receptor 1
(IC50: 0.49 µmol/L) and also blocks the related
tyrosine kinases PDGF ß-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 µmol/L,
respectively). In concentrations up to 10 µmol/L, PTK787 does not
inhibit kinases from other families, including FGFR1, c-Met, Tie2,
EGFR, c-Src, v-Abl, and PKC-
. The dihydrochloride salt of PTK787 was
used for all experiments.
CGP 57148 and CGP 53716 are selective inhibitors of the kinase activity of PDGF ß-receptors (IC50s: 0.3 and 0.25 µmol/L, respectively).and v-Abl (IC50s: 0.2, 0.38, and 1.2 µmol/L, respectively) and do not inhibit phosphorylation by other kinases, including VEGF receptors or PKCs in concentrations greater than 20 µmol/L.18-20
PTK787 Inhibits Retinal Neovascularization in Mice with Ischemic Retinopathy
Figure 1, A and B
, shows the
appearance of normal griffonia simplicifolia lectin-stained retinal
vessels in nonischemic P17 mice. In P17 mice with ischemic retinopathy,
there is a marked increase in the area of endothelial cell staining
throughout the retina with large clumps of cells constituting NV on the
retinal surface (Figure 1, C and D
; Figure 2E
, columns 2 versus 1). In
contrast, P17 mice with ischemic retinopathy treated twice a day by
gavage with 25 mg/kg of PTK787 showed a dramatic decrease in the area
of endothelial cell staining within the retina (Figure 1E)
. There were
vessels in the innermost part of the retina inside the inner limiting
membrane adjacent to the vitreous cavity, the region normally occupied
by the superficial capillary bed, but there were no vessels on the
surface of the retina and the deep capillary beds were absent. The
avascular appearance of the retina was very similar to that of mice
with ischemic retinopathy treated with 600 mg/kg/day of PKC412, a
partially selective kinase inhibitor that blocks phosphorylation by
VEGF and PDGF receptors and several isoforms of PKC.15
Like retinas from mice treated with PKC412, high magnification of
retinas from mice treated with PTK787 showed no identifiable
endothelial cells above the internal limiting membrane on the surface
of the retina (Figure 1F)
. All retinal sections from mice with ischemic
retinopathy treated with 50 mg/kg/day of PTK787 had no endothelial
cells above the internal limiting membrane, indicating that there was
complete inhibition of retinal NV. To assess the effect on retinal
vascular development as well as retinal NV, we quantified the total
area of endothelial cell staining in the retina in
PTK787-treated and control mice by image analysis. There was a dramatic
decrease in retinal endothelial cells in mice with ischemic retinopathy
treated with 25 mg/kg twice a day (120 µmoles/kg/day) of PTK787
compared to vehicle-treated mice (Figure 2E
, columns 5 and 2,
respectively), and a significant decrease from nonischemic mice (Figure 2E
, columns 5 and 1, respectively).
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, arrows) and decreased staining in the regions of the deep capillary
beds compared to vehicle-treated mice (Figure 1, G and C
,
respectively). Image analysis demonstrated a statistically significant
difference in the area of endothelial cell staining (Figure 2E
, columns
4 versus 2). The retinas of mice with ischemic retinopathy
treated orally twice a day with 0.25 mg/kg had the appearance of less
endothelial cell staining than retinas of vehicle-treated mice (Figure 1, G
versus C). The difference was not statistically
significant by paired t-test, but analysis of variance
demonstrated a statistically significant dose-dependant decrease in
endothelial cell area for the 3 doses of PTK787 compared to vehicle
alone (P < 0.001; Figure 2E
).
At 120 µmoles/kg/day, a dose equivalent to the highest dose of PTK787
used, CGP 57148 (Figure 2C)
and CGP 53716 (Figure 2D)
, two selective
PDGF receptor tyrosine kinase inhibitors (PDGF-RTKI), caused no
apparent decrease in NV in mice with ischemic retinopathy compared to
vehicle-treated mice (Figure 2A)
. This was confirmed by quantitative
analysis in mice treated with CGP 57148 (Figure 2E
, column 7
versus 2). At 120 µmoles/kg/day, genestein, a nonspecific
tyrosine kinase inhibitor, caused a small, statistically significant
decrease in the area of endothelial cell staining in the retinas of
mice with ischemic retinopathy (Figure 2, B
versus A and
Figure 2E
, columns 6 versus 2).
PTK787 Inhibits Retinal Vascular Development
Treatment of neonatal mice with PKC412, the inhibitor of VEGF and
PDGF receptors and PKC, results in inhibition of retinal vascular
development.15
To determine whether PTK787 inhibits
retinal vascular development, neonatal mice were treated with
subcutaneous injections of 10 mg/kg of PTK787 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 had almost reached the peripheral edge of
retina, but in PTK787-treated mice they had only extended about halfway
to the periphery (Figure 3, A and B)
. At
P10 in control mice, the superficial capillary bed was complete and
extended all the way to the peripheral edge of the retina, and the deep
capillary bed was partially developed. But in PTK787-treated mice, the
superficial capillary bed had not yet reached the edge of the retina
(Figure 3, C and D)
. Measurement of the distance from the optic nerve
to the developing edge of retinal vessels at 7 and 10 days showed a
highly significant decrease at both time points in PTK787-treated mice
compared to controls (Figure 4)
.
Therefore, PTK787, like PKC412, inhibits retinal vascular development
as well as pathological retinal NV. However, it is interesting to note
that at the doses used, both of these drugs delay, but do not
permanently prevent, development of the superficial capillary bed,
which occurs by vasculogenesis. On the other hand, a 2.5-fold higher
dose of PTK787, given between P7 and P17, completely inhibits
development of the intermediate and deep capillary beds (Figure 1, E and F)
, which occurs by the process of angiogenesis. Additional studies
are needed to determine whether these differences are dose- and/or
timing-related, or whether they indicate that angiogenesis is more
completely dependent on VEGF signaling than is vasculogenesis.
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Transgenic mice in which the rhodopsin promoter drives
retina-specific expression of VEGF (rho/VEGF mice)
develop widespread NV that originates from retinal vessels, extends
through the outer nuclear layer, and grows in the subretinal
space.17
In these mice, the expression of VEGF is turned
on in photoreceptors around P6 or P7, increases to a steady-state level
over a few days, and is sustained for at least several months, the
longest time points yet examined. At 3 weeks of age, there is extensive
NV in the subretinal space that is easily quantitated on retinal whole
mounts.16
Administration of 25 mg/kg once a day (60
µmoles/kg/day) of PTK787 between 7 and 21 days resulted in complete
absence of NV in the subretinal space of rho/VEGF mice
(Figure 5)
. Focusing on the outer surface
of the retina, which corresponds to the subretinal space, there was no
NV in PTK787-treated mice (Figure 5B)
, whereas in vehicle-treated mice,
there were numerous areas of NV (Figure 5, A and C)
. Focusing at the
level of the deep capillary bed, some abortive buds could be seen
extending from retinal vessels in PTK787-treated mice (Figure 5D)
. This
indicates that PTK787 is an extremely effective inhibitor of
VEGF-induced NV in the retina.
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Adult mice were treated twice a day for 5 days with 25 mg/kg (120
µmoles/kg/day) of PTK787, a dose and duration of dosing that
completely inhibit retinal NV in oxygen-induced ischemic retinopathy
and a greater dose than that shown to inhibit development of the deep
retinal capillary bed. The lectin-stained retinal vessels of
PTK787-treated adult mice (Figure 6B)
appeared normal and identical to the retinal vessels of control mice
(Figure 6A)
. Image analysis demonstrated no difference in the total
area of endothelial cell staining in the retinas of treated
versus control mice (Figure 6E)
. Retinas from treated mice
stained with H&E were also completely normal and identical in
appearance to retinas from control mice (Figure 6, D
versus
C). This suggests that treatment with PTK787 for 5 days is not toxic to
endothelial cells of mature vessels or to retinal neurons.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently demonstrated that it is possible to achieve complete inhibition of retinal NV in the murine model of oxygen-induced ischemic retinopathy by oral administration of a partially selective kinase inhibitor that blocks phosphorylation by VEGF and PDGF receptors, and by serine/threonine kinases of the PKC family.15 In the present study, we found complete inhibition of retinal NV with a different kinase inhibitor that blocks phosphorylation by VEGF and PDGF receptors, but not PKC. Two drugs that selectively block PDGF receptor kinase activity, but not VEGF receptor kinase or PKC, had no significant effects on retinal NV. By process of elimination, it appears that inhibition of VEGF receptor kinase is the activity responsible for the dramatic inhibition of retinal NV in this model of ischemic retinopathy.
A new model of retinal NV in transgenic mice with photoreceptor-specific expression of VEGF16,17 differs from the ischemic retinopathy model in that VEGF is the only known stimulus and its secretion is high and sustained, so there is no spontaneous involution of the NV. Oral administration of PTK787 resulted in complete inhibition of NV in the transgenic model, confirming that this drug is a very effective inhibitor of VEGF signaling in the retina. The ability of PTK787 or PKC41215 to block VEGF signaling in the ischemic retinopathy model also resulted in complete inhibition of retinal NV. This suggests that despite possible contributions from other growth factors, blockade of VEGF receptor signaling is sufficient to completely inhibit retinal NV. The relative lack of efficacy of intraocular injections of VEGF receptor/IgG chimeric proteins12 or anti-sense VEGF oligomers13 could be due to delivery problems, or it may be that inhibition of VEGF receptor kinase activity is a more effective way to block VEGF signaling than these two approaches aimed at decreasing the amount of VEGF available to bind to receptors.
Blockade of VEGF signaling with PTK787 or PKC41215 in neonatal mice inhibits retinal vascular development. This is not surprising in view of previous data demonstrating that VEGF signaling is necessary for the survival of developing retinal vessels.2 Considering this and our demonstration that retinal vascular development is perturbed in neonatal mice treated with either of two potent VEGF receptor kinase inhibitors, it is likely that any drug that effectively blocks VEGF signaling will have a deleterious effect on retinal vascular development. The lack of a similar effect in the neonatal mouse model of ischemic retinopathy with other VEGF antagonists12,13 suggests that with those approaches, blockade of VEGF signaling was incomplete. Although there may be a narrow therapeutic window within which it is possible to completely or almost completely inhibit retinal NV without damaging developing retinal vessels, it would be hazardous to treat infants with ROP with highly effective VEGF inhibitors. However, mature retinal vessels are much less dependent on VEGF as a survival factor,2 and in support of this, we saw no evidence of damage to retinal vessels in adult mice treated with PTK787 or PKC412 for 5 days. This suggests that PTK787, PKC412, or other potent VEGF receptor kinase inhibitors could potentially be useful for treatment of proliferative diabetic retinopathy or other ischemic retinopathies in patients with mature retinas. Clinical trials to investigate safety and efficacy of VEGF kinase inhibitors in patients with proliferative diabetic retinopathy are being planned.
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Footnotes |
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Supported by U. S. Public Health Service grant EY05951 and core grant P30EY1765 from the National Eye Institute, a Lew R. Wasserman Merit Award (to P. A. C.) and an unrestricted grant from Research to Prevent Blindness, Inc., 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. C. 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.
H. Ozaki’s current address: Fukuoka University School of Medicine, Fukuoka, Japan.
M.-S. Seo’s current address: Chonnam National University Medical School and Hospital, Kwangju, Korea.
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.
Accepted for publication October 6, 1999.
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References |
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-v ß-3 inhibit retinal neovascularization in a murine model. Lab Invest 1996, 75:563-573[Medline]
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