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Angiostatin Inhibits and Regresses Corneal Neovascularization
Balamurali K. Ambati, MD;
Antonia M. Joussen, MD;
Jayakrishna Ambati, MD;
Yasufumi Moromizato, MD;
Chandan Guha, MBBS, PhD;
Kashi Javaherian, MD;
Stephen Gillies, PhD;
Michael S. O'Reilly, MD;
Anthony P. Adamis, MD
Arch Ophthalmol. 2002;120:1063-1068.
ABSTRACT
| |
Objective To determine the ability of angiostatin and the angiostatin-producing
low-metastatic (LM) clone of Lewis lung carcinoma (LLC) to inhibit and regress
corneal neovascularization, as compared with the non–angiostatin-producing
high-metastatic (HM) clone.
Methods Three groups of C57BL6/J mice underwent chemical and mechanical denudation
of corneal and limbal epithelium. One group remained tumor free while the
other 2 were implanted with LLC cells (either the HM or LM clones) subcutaneously
the day before, 2 weeks after, or 4 weeks after denudation. Corneas were harvested
2 weeks after tumor implantation (at 2, 4, and 6 weeks after denudation for
tumor-free mice). Neovascularization was quantified by CD31 immunostaining.
In a second experiment, recombinant angiostatin was delivered continuously
for 2 weeks via an osmotic pump in mice with established corneal neovascularization.
Results The mean percentages of neovascularized corneal area in mice 2 weeks
after LM-LLC implantation were 4.6%, 3.7%, and 37.0%, at 2, 4, and 6 weeks
after scraping, respectively. In contrast, in the mice implanted with HM-LLC,
the corresponding values were 45.4% (P = .01), 90.1%
(P = .03), and 80.3% (P
= .005). For tumor-free mice, the corresponding values were 62.0% (P = .003), 68.9% (P = .03), and 59.3% (P = .06). Mice implanted with angiostatin pumps had a 37.7%
neovascularized corneal area 2 weeks after implantation and 4 weeks after
scraping while mice implanted with sham pumps had 60.5% (P = .007).
Conclusion Angiostatin inhibits and regresses corneal neovascularization induced
by mechanical and alkali corneal injury.
Clinical Relevance This appears to be the first evidence of biologically induced regression
of corneal neovascularization, and the first direct demonstration of angiostatin-induced
regression of neovascularization in any tissue.
INTRODUCTION
CORNEAL neovascularization is a central feature in the pathogenesis
of many blinding corneal disorders and a major sight-threatening complication
in corneal infections, chemical injury, and following keratoplasty, in which
neovascularization adversely affects corneal graft survival.1
Antiangiogenic molecules have been shown to inhibit corneal neovascularization.
However, no biologic agent has been shown to regress established corneal neovascularization,
a more clinically relevant end point. Thermal laser or photodynamic therapy
induces only temporary closure of new vessels2
and does not address the underlying biological mechanisms of neovascularization.
Angiostatin, an endogenous cleavage fragment of plasminogen, is secreted
by a form of Lewis lung carcinoma (LLC) and inhibits basic fibroblast growth
factor–induced corneal neovascularization in mice.3
Its antiangiogenic potential has been verified in numerous tumor models.4 We investigated the potential of an angiostatin-producing
tumor to inhibit and regress corneal neovascularization in a pathophysiologically
relevant murine model. In a confirmatory experiment, we also investigated
the ability of pure recombinant angiostatin delivered systemically via an
osmotic pump to regress corneal neovascularization in the same model.
DESIGN AND METHODS
All animal experiments were approved by the Massachusetts Eye and Ear
Infirmary (Boston) animal care committee and conformed to the Association
for Research in Vision and Ophthalmology guidelines for animal use. Male C57BL/6J
mice (Jackson Labs, Bar Harbor, Me) were anesthetized by intramuscular injection
of 50 mg/kg of ketamine hydrochloride and 10 mg/kg of xylazine. Animals were
killed by a lethal dose of pentobarbital (150 mg/kg).
EXPERIMENTAL DESIGN
Three groups of mice underwent corneal injury. One group (referred to
hereafter as control) remained tumor free and the other 2 were implanted with
LLC tumor cells (either the high-metastatic [HM] or low-metastatic [LM] clones)
subcutaneously the day before, 2 weeks after, or 4 weeks after injury by an
investigator masked to the clone type. Corneas were harvested 2 weeks after
tumor implantation (at 2, 4, and 6 weeks after injury for tumor-free mice).
In a separate regression experiment, angiostatin pumps were implanted 2 weeks
after corneal injury. Corneas were harvested 2 weeks after implantation and
compared with corneas from control mice (implanted with pumps containing phosphate-buffered
saline [PBS] alone) harvested 4 weeks after corneal injury. Therefore, the
time of treatment with angiostatin was 2 weeks in all experimental groups.
Neovascularization was quantified in all corneas by immunostaining, using
an image analyzer by a masked investigator.
MODEL OF CORNEAL NEOVASCULARIZATION
Topical proparacaine and 2 µL of 0.15M sodium hydroxide were applied
to the right cornea of each mouse. The corneal and limbal epithelia were removed
using a Tooke corneal knife (Arista Surgical Supply, New York, NY) in a rotary
motion parallel to the limbus. Erythromycin ophthalmic ointment was instilled
immediately following epithelial denudation.
MODEL OF LLC IMPLANTATION
Low-metastatic LLC tumors were passaged in vivo3
and HM-LLC cells were passaged in vitro.5 Recipient
mice had their backs shaved, and the recipient site was cleansed with povidone-iodine
and ethanol. The subcutaneous dorsa of the mice in the proximal midline were
injected with 0.1 mL containing 106 cells of the respective tumor
cell lines.
ANGIOSTATIN PUMP IMPLANTATION
Murine angiostatin was produced as a fusion protein with the murine
immunoglobulin 2a Fc fragment (mFc-m-angiostatin) in a murine myeloma cell
line as described previously.6 The protein
was diluted in PBS, filtered through a Millipore filter (Millipore, Bedford,
Mass), and stored at -20°C until used. The purity of the mFc-m-angiostatin
ranged between 90% and 95% (data not shown). The murine recombinant angiostatin
was continuously delivered with a mini-osmotic pump. Mini-osmotic pumps with
a pump rate of 1.0 µL per hour were implanted into the intraperitoneal
cavity 2 weeks after limbal injury (n = 6 animals). The total delivered dose
of angiostatin was 10 mg/kg per day. After 8 days, the pumps were exchanged
and animals were killed 2 weeks after the first pump was implanted. Control
animals received pumps delivering equal amounts of PBS (n = 7 animals).
LABELING OF CORNEAL NEOVASCULARIZATION
Immunohistochemical staining for vascular endothelial cells was performed
on corneal flat mounts. Fresh corneas were dissected, rinsed in PBS for 30
minutes, and fixed in 100% acetone for 20 minutes. After washing in PBS, nonspecific
binding was blocked with 0.1M PBS, 2% albumin for 1 hour at room temperature.
Incubation with fluorescein isothiocyanate conjugated–coupled monoclonal
antimouse CD31 antibody at a concentration of 1:500 in 0.1 M of PBS, 2% albumin
at 4°C overnight was followed by subsequent washes in PBS at room temperature.
Corneas were mounted with Gelmount (Biomeda Inc; San Francisco, Calif), an
antifading agent, and visualized with a fluorescent microscope.
QUANTIFICATION OF CORNEAL NEOVASCULARIZATION
Digital quantification of corneal neovascularization has been previously
described.7-8 Images of the corneal
vasculature were captured using a CD-330 charge-coupled device camera attached
to a Leica MZ FLIII fluorescent microscope (Leica Microsystems Inc, Deerfield,
Ill). The images were analyzed using Openlab software (Improvision Inc, Lexington,
Mass), resolved at 624 x 480 pixels, and converted to tagged information
file format files. The neovascularization was quantified by setting a threshold
level of fluorescence above which only vessels were captured. The entire mounted
cornea was analyzed to minimize sampling bias. The quantification of the neovascularization
was performed in masked fashion. The total corneal area was outlined using
the innermost vessel of the limbal arcade as the border. The total area of
neovascularization was then normalized to the total corneal area and the percentage
of the cornea covered by vessels calculated.
TUMOR BURDEN
All euthanized animals that had been implanted with a tumor underwent
autopsy to identify metastases. The weights of the animals were measured using
an AE 200 microbalance (Mettler, Toledo, Ohio).
STATISTICS
Differences in areas of corneal neovascularization were analyzed with
the Wilcoxon (Mann-Whitney) test and the Kruskal-Wallis test.
RESULTS
There were no significant differences in corneal neovascularization
between corresponding groups of control mice and mice implanted with HM-LLC
(all P values >0.1). There was no significant spontaneous
regression of corneal neovascularization in tumor-free mice (P = .87).
PERCENTAGE AREA OF CORNEAL NEOVASCULARIZATION
Inhibition Groups
Mean percentages of neovascularized cornea area in mice 2 weeks after
corneal scraping and implantation are shown in Figure 1. The mean percentages of neovascularized corneal area in
mice 2 weeks after denudation in control mice, mice implanted with LM-LLC,
and mice implanted with HM-LLC were 62.0%, 4.6%, and 45.4%, respectively.
Thus, mice implanted with LM-LLC had 89.9% less neovascularized corneal area
than those implanted with HM-LLC (P = .01) and 92.6%
less neovascularized corneal area than control mice (P
= .003).
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Figure 1. Mean fraction of corneal neovascularization
(±SEM) 2 weeks after tumor implantation and corneal scraping in control
mice (n = 7), mice implanted with low-metastatic Lewis lung carcinoma (LM-LLC)
(n = 5), and mice implanted with high-metastatic (HM) LLC (n = 4).
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Regression Groups
Mean percentages of neovascularized corneal area in mice 2 weeks after
tumor implantation and 4 or 6 weeks after corneal scraping are shown in Figure 2. The mean percentages of neovascularized
corneal area in mice 4 weeks after denudation in control mice, mice implanted
with LM-LLC, and mice implanted with HM-LLC were 68.9%, 3.7%, and 90.1%, respectively.
Thus, mice implanted with LM-LLC had 95.9% less neovascularized corneal area
than those implanted with HM-LLC (P = .03) and 94.6%
less neovascularized corneal area than control mice (P
= .03).
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Figure 2. Mean fraction of corneal neovascularization
(±SEM) 2 weeks after tumor implantation and 4 weeks after corneal scraping
in control mice (solid) (n = 3), mice implanted with low-metastatic Lewis
lung carcinoma (LM-LLC) (n = 3), and mice implanted with high-metastatic LLC
(HM-LLC) (n = 3), and 6 weeks after corneal scraping in control mice (n =
7), mice implanted with LM-LLC (n = 4), and mice implanted with HM-LLC (n
= 4).
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The mean percentages of neovascularized corneal area in mice 6 weeks
after denudation in control mice, mice implanted with LM-LLC, and mice implanted
with HM-LLC were 59.3%, 37.0%, and 80.3%, respectively. Thus, mice implanted
with LM-LLC had 53.9% less neovascularized corneal area at 6 weeks after corneal
scraping than those implanted with HM-LLC (P = .005)
and 37.6% less neovascularized corneal area than control mice (P = .06). Representative photographs are shown in Figure 3.
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Figure 3. Photographs of corneal flat mounts
of mouse eyes 6 weeks after corneal injury stained with fluorescein isothiocyanate
conjugated–coupled monoclonal murine antiCD31 antibody. A, Control eye
(no tumor implanted) (original magnification x10). B, Eye from mouse
2 weeks after low-metastatic Lewis lung carcinoma (LLC) implantation (original
magnification x10). C, Eye from mouse 2 weeks after high-metastatic
LLC implantation (original magnification x10). D-F, High-magnification
(x40) views of A, B, and C, respectively.
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PERCENTAGE AREA OF CORNEAL NEOVASCULARIZATION: ANGIOSTATIN PUMP VS
CONTROL PUMP
Mean percentages of neovascularized corneal area in mice 2 weeks after
pump implantation and 4 weeks after corneal scraping are shown in Figure 4. Mice with angiostatin pumps inserted
had 37.7% less neovascularized corneal area than mice with sham pumps (P = .007).
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Figure 4. Mean fraction of corneal neovascularization
(±SEM) 2 weeks after pump implantation and 4 weeks after corneal scraping
in sham pump (n = 7), and angiostatin pump (n = 6).
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TUMOR BURDEN
No animal that had been implanted with LM-LLC had visceral metastases,
whereas all animals implanted with HM-LLC were noted to have liver and/or
lung metastases. The mean ± SD weights of LM-LLC mice at 2, 4, and
6 weeks after implantation were 16.8 ± 0.6 g, 19.1 ± 0.3 g,
and 21.8 ± 0.6 g, respectively. The mean ± SD weights of HM-LLC
mice at 2, 4, and 6 weeks after implantation were 16.5 ± 0.5 g, 19.3
± 0.3 g, and 22.0 ± 0.4 g, respectively (all P values >0.1).
COMMENT
These data demonstrate that the LM clone of LLC, an angiostatin-producing
tumor, can inhibit and regress corneal neovascularization induced by corneal
injury secondary to mechanical and alkali trauma. In contrast, HM-LLC, a spontaneous
variant of the LM-LLC tumor that is unable to generate angiostatin, was unable
to inhibit angiogenesis or regress neovascularization. These data were confirmed
in experiments showing the regression of corneal neovascularization induced
by recombinant angiostatin delivered systemically via an osmotic pump. To
our knowledge, this is the first report of biologically induced regression
of corneal neovascularization as well as the first direct demonstration in
any tissue of regression of blood vessels likely mediated by angiostatin.
The results indicate near complete inhibition of corneal neovascularization
by LM-LLC (in accordance with previous data) and near-complete regression
of corneal neovascularization when LM-LLC was implanted 2 weeks after corneal
injury. The regressive effect was substantially less when LM-LLC was implanted
4 weeks after injury. This may indicate that the more mature vessels are less
amenable to the ability of angiostatin to induce regression or they may require
further exposure to angiostatin to regress. This was not addressed in our
study, as mice generally died of tumor burden from LM-LLC between 18 and 24
days, and supplies of recombinant angiostatin pumps did not permit longer-term
or varied dose-effect experiments. The mechanisms of the regression of the
new vessels are not fully characterized. Down-regulation of vascular endothelial
growth factor can induce the regression of capillaries not covered by pericytes
in the corpus luteum of ovaries, while increased levels of angiopoietin-2
destabilize mature capillaries and induce their regression.9
It would be valuable to determine to what extent angiostatin is capable of
the 2 effects.
Angiostatin inhibits the development of basic fibroblast growth factor–
and vascular endothelial growth factor–induced corneal neovascularization
in mice.3 Other studies have shown that thalidomide,
curcumin, integrin antagonists, cyclooxygenase inhibitors, prolactin, octreotide,
herbal extracts, matrix metalloproteinase inhibitors, angiostatic steroids,
thrombospondin-2, kringle 1-3, and beta-cyclodextrin tetradecasulfate all
exert inhibitory effects on the development of corneal neovascularization
induced by various methods.10-23
Our study demonstrates the ability of angiostatin to inhibit neovascularization
in a clinically relevant model of mechanical and chemical corneal trauma.
The method of neovascularization induction affects pharmacological efficacy,
eg, integrin antagonists inhibit basic fibroblast growth factor–induced
corneal neovascularization but not that caused by chemical injury.10 Moreover, this study demonstrates for the first time,
to our knowledge, the biologically induced regression of corneal neovascularization.
The only other interventions demonstrated to affect established corneal neovascularization
have been laser photocoagulation, fine-needle diathermy, and photodynamic
therapy.24-28
However, these methods do not induce biological vessel regression, as was
observed with angiostatin. Angiostatin's ability to inhibit blood vessels
on the basis of tumor regression and endothelial apoptosis has been demonstrated
in culture4, 28; our study provides
a direct in vivo correlate.
The antiangiogenic effect observed in animals implanted with LM-LLC,
as opposed to HM-LLC, has been localized to angiostatin. O'Reilly et al3 purified the serum and urine of LM-LLC and HM-LLC
mice with reverse-phase chromatography and gel electrophoresis and assayed
different fractions for inhibition of endothelial proliferation; inhibitory
activity corresponded to angiostatin. Dong et al29
performed fast protein liquid chromatography and Western blot analysis and
found that the antiangiogenic activity of serum from LM-LLC mice localized
to angiostatin and also found that a monoclonal antibody against angiostatin
blocked the antiangiogenic effect (measured by an assay of endothelial cell
proliferation) of LM-LLC serum in culture. O'Reilly et al30
found that blocking the production of angiostatin by LM-LLC cells in culture
using antibodies to gelatinase A (required for angiostatin production) eliminated
the antiangiogenic effect of these cells, and that only fractions generated
by gelatinase A that contained angiostatin had antiangiogenic effects (measured
by ability to inhibit endothelial cell proliferation).
The level of regression in tumor-implanted mice was far greater than
that induced by the angiostatin pump. This may be owing to several factors.
The concentration of angiostatin produced by the tumor may be much higher
than that delivered by the pump, although it is not possible to quantitate
serum or urine levels of angiostatin because of lack of commercial assays.
The concentration of angiostatin in the tumor model is also variable and likely
steadily rises as the tumor grows, and this variability may enhance the regressive
effect.
In our study, the tumor burden was not significantly different between
the LM-LLC and the HM-LLC groups. The HM-LLC clone arose as a spontaneous
variant of the LM-LLC clone in mice and it did not produce angiostatin in
vitro or in vivo. Therefore, the antiangiogenic effects observed herein can
very likely be attributed to angiostatin. The implantable pump study results
are consistent with this conclusion.
Digital quantification of corneal neovascularization, described previously7-8 using flat-mounted corneas, can potentially
be confounded by the 3-dimensional nature of specimens and the density of
blood vessels. The dehydration of corneas prior to analysis and the use of
a microscope with a good depth of field minimize the effect of 3-dimensionality.
This is especially true in the very thin mouse cornea. The density of blood
vessels in corneas with a high degree of neovascularization is potentially
a problem because individual vessels sometimes cannot be discerned, leading
to an overestimation of vascularization as areas of clear cornea in between
are not resolvable. This is countered by the effect of underestimation of
overlapping vessels. Previous investigators7-8
have found the magnitude of these errors to be small and not to affect the
consistency among samples.
In summary, angiostatin inhibits and regresses the corneal neovascularization
induced by mechanical and alkali corneal injury. This finding could advance
the management of blinding disorders, such as Stevens-Johnson syndrome, cicatricial
pemphigoid, corneal allograft rejection, and corneal injury from infection,
trauma, or alkali. The potential of topical or sustained-release forms of
angiostatin with distribution constrained to the eye should be a next line
of investigation. Topical forms of angiostatin have already been used in the
rat and rabbit.31-32 Gene transfer
methods generating angiostatin have been developed to halt angiogenesis in
mouse tumor models,33-34 and naked
plasmids have been shown to be taken up by corneal epithelium.35
Future research should also determine the effect of recombinant angiostatin
in this model and the molecular interactions among angiostatin, inflammatory
phenomena, and neovascularization. This model can also be extended to neovascularization
in other ocular tissues, including the iris, retina, and choroid.
AUTHOR INFORMATION
Submitted for publication October 16, 2001; final revision received
April 1, 2002; accepted April 24, 2002.
This work was supported by the American Ophthalmological Society-Knapp
Testimonial Fund, Chicago, Ill (Dr J. Ambati), a Foundation Fighting Blindness
Career Development Award, Owings Mills, Md (Dr J. Ambati), Deutsche Forschungsgemeinschaft
Jo 324/2-1, Bonn, Germany, the Massachusetts Lions Eye Research Fund, Boston
(Dr Adamis), and the Roberta Siegel Research Fund, Boston (Dr Adamis).
Corresponding author and reprints: Anthony P. Adamis, MD, Department
of Ophthalmology, Massachusetts Eye & Ear Infirmary, 243 Charles St, Boston,
MA 02114 (e-mail: tony_adamis{at}meei.harvard.edu).
From the Department of Ophthalmology, Massachusetts Eye and Ear Infirmary,
(Drs B. K. Ambati, Joussen, J. Ambati, Moromizato, and Adamis); Laboratory
for Surgical Research, Children's Hospital (Drs Joussen, Moromizato, Javaherian,
and Adamis), Harvard Medical School, Boston; Department of Radiation Oncology,
Albert Einstein College of Medicine, Bronx, NY (Dr Guha); Lexigen Pharmaceuticals,
Lexington, Mass (Dr Gillies); Division of Radiation Oncology, University of
Texas M. D. Anderson Cancer Center, Houston (Dr O'Reilly).
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