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1 From the Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, Nashville, Tennessee; and 2 Alcon Laboratories, Inc., Fort Worth, Texas.
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Abstract |
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METHODS. At birth, rats were placed in an atmosphere of varying oxygen that produces retinal neovascular changes that approximate human ROP. The rats then received intravitreal injections of either anecortave acetate or vehicle at varying times, and all were subsequently placed in room air. Retinas were assessed for plasminogen activator inhibitor (PAI)-1 mRNA level by RNase protection assay at 1, 2, and 3 days after injection and for normal and abnormal blood vessel growth 3 days later.
RESULTS. A significant reduction in the severity of abnormal retinal neovascularization was observed in the steroid-treated eyes compared with vehicle-injected eyes in ROP rats, yet the extent of normal total retinal vascular area was not significantly different. The drug had no effect on either retinal vascular area or neovascularization when tested in room air–raised control rats. Drug-injected eyes demonstrated a six- to ninefold increase in PAI-1 mRNA at 1 to 3 days after injection.
CONCLUSIONS. This study represents the first therapeutic effect of an angiostatic steroid in an animal model of neovascular retinopathy. Additionally, the induction of PAI-1 indicates a mechanism of action for this class of compounds, and this is a novel finding in vivo. Because anecortave acetate significantly inhibited pathologic retinal angiogenesis in this model, while not significantly affecting normal intraretinal vessels, it holds therapeutic potential for a number of human ocular conditions in which angiogenesis plays a critical pathologic role.
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Introduction |
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In 1985, a new class of steroids that inhibits angiogenesis, yet has little glucocorticoid (anti-inflammatory) or mineralocorticoid (salt-retaining) activity, was introduced.13 These agents, which are called angiostatic steroids, have since proven effective in inhibiting angiogenesis in a variety of systems, including chick chorioallantoic membrane,13 14 15 rat mammary carcinoma,16 rabbit cornea,15 17 18 rat cornea,19 and mouse intraocular tumors.20 The mechanism of inhibition of angiogenesis has yet to be identified in any of these systems.
Retinopathy of prematurity (ROP) is a pathologic condition of the retina in which abnormal angiogenesis (called retinal neovascularization, in this context) can lead to devastating consequences as a result of vitreal bleeding and tractional retinal detachment. The increasing incidence of ROP is linked to the implementation of new neonatal care technologies that enhance the survival of very low birth weight infants21 and the continued need for supplemental oxygen to maintain arterial oxygen tension in an appropriate range. Our understanding of the course of ROP has been increased with the advent of several innovations, including an international ROP classification system,22 a consensus protocol for screening neonatal intensive care units.23 The prevalence of severe ROP has been reduced by a recommended course of surgical intervention to halt the disease.24 However, because the surgical intervention yields limited functional success,24 25 and because the procedures are themselves traumatic and invasive, there is a need for a preventive therapy.
We have tested the capacity of the angiostatic steroid, anecortave acetate, to inhibit retinal neovascularization in a rat model of ROP. The model used was established in our laboratory several years ago26 and it has been reproduced and used in published studies by several other investigators.27 28 The model has the attractive features that the pathology is extensively characterized; its severity is consistent between experiments, but it can be manipulated by altering critical experimental variables; the conditions are clinically relevant; and, unlike other published rodent models, the neovascular tufts appear in a retinal pattern mimicking that occurring in premature infants with ROP.
To our knowledge, this report is the first demonstration of a therapeutic influence of an angiostatic steroid on retinal angiogenesis. In addition, we have initiated investigations into the mechanism of the angiostatic influence of anecortave acetate. We have focused on the ability of the compound to induce plasminogen activator inhibitor (PAI)-1 mRNA. This endogenous protease inhibitor counterbalances the action of plasminogen activator—an upstream initiator of proteolysis of the extracellular matrix. The induction of PAI-1 by angiostatic steroids has been demonstrated in vitro,29 but to date this influence has not been identified in animal studies.
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Methods |
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Intravitreal Injections
Rats were anesthetized with a 40 mg/kg intraperitoneal injection
of ketamine along with topical application of 0.5% proparacaine to the
eye. The fused eyelids were opened and temporally canthotomized, when
necessary, and then local anesthetic was reapplied, followed by 1%
mydriacyl. While proptosing with gentle pressure on the lids, the globe
was penetrated within 0.5 ± 0.15 mm posterior to the ora serrata
(measured subsequently, after retinal flat-mounting) with a 28-gauge
needle. The needle was immediately removed and a blunt-tipped, 30-gauge
10 µl syringe (Hamilton Co., Reno, NV) was inserted through the
existing hole. Contact with the posterior surface of the lens was
avoided by maintaining a steep angle, which delivered the injection
bolus near the trunk of the hyaloid artery, just above the posterior
pole of the retina. After the needle was removed, topical antibiotic
cocktail (neomycin and polymyxin B sulfates and gramicidin) was applied
to the point of entry. Noninjected eyes also were treated with topical
proparacaine and antibiotic to control for the potential of these
agents to influence retinal vessel growth. Vitreous hemorrhage occurred
immediately after injection in two cases, and these rats were
eliminated from the study and replaced with alternates.
Drug Treatment Protocol
Anecortave acetate
[4,9(11)-Pregnadien-17
,21-diol-3,20-dione-21-acetate] (Fig. 1)
was obtained from Alcon Laboratories, Inc. (Fort Worth, TX). The
experimental protocol for these studies is presented in Figure 2
. At 14 days, immediately after removal of the VOE rats from the
exposure chamber, rats in both the RA and the VOE groups received an
intravitreal injection in the left eye. Half of the rats
(n = 12) received 5 µl of a 10% suspension of
anecortave acetate and the remainder (n = 12) received
5 µl of vehicle (70:20:10 by volume, polyethylene glycol: phosphate
buffered saline: EtOH). After 2 days in room air (Day 16), the
injection protocol was replicated in the right eye of each animal
(i.e., same agent and volume as in the left eye). The dosage of steroid
used was based on dose/response optimization trials previously
performed in our laboratory (data not shown).
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To determine the effect of anecortave acetate on the normal development of intraretinal blood vessels, a limited trial was conducted in rats raised in room air. Seven-day-old rats were chosen for this experiment because the growth of retinal blood vessels from the optic nerve head, where the process starts, to the far retinal periphery where it ends, is about half-complete at this age. Any inhibition of the process over the next several days is easily measured. At 7 days of age, 5 µl of 10% anecortave acetate was injected into the left eye and 5 µl of vehicle into the right (n = 8). Additional 7-day-olds were left with no injection (n = 5). These rats were killed at 10 days of age, a day or two before the time when the first retinal vessels normally reach the ora serrata.
Effect on Retinal Vascular Development
After enucleation and dissection of the retinas in 10% neutral
buffered formalin, the retinas were flattened and stored overnight at
4°C. Retinas were then processed for adenosine diphosphatase (ADPase)
histochemistry using a modification31
of the method of
McLeod and coworkers.32
This process primarily stains
vascular endothelia and their stem cells in rats of this
age.31
Images of ADPase-stained retinas were digitized and captured (Imagegrabber 2.0; Neotech, Hampshire, UK). Retinal areas containing blood vessels were then traced on the computer monitor face with an interactive stylus pen (FT Data Systems, Stanton, CA). The operator was masked with respect to the treatment. The area within the trace was calculated with image analysis software (Enhance 3.0; Microfrontier, Des Moines, IA) and is reported in mm2. Measurements of this parameter were recorded and statistically significant differences between the treatment groups were determined by analysis of variance with Scheffé’s post hoc procedure.33 Data normality was determined by the D’Agostino test.34 Statistical analysis of the data from rats killed at both 10 and 18 days of age was conducted in this manner.
Effect on Retinal Neovascularization
To determine the effect of anecortave acetate on retinal
neovascularization, the degree of vascular pathology was assessed in
the flattened, ADPase-stained retinas. As the retinas were flattened,
care was taken to ensure that each of the four quadrants was nearly
equal in size. Each of these quadrants was divided into three
theoretical clock hours. Each clock hour that was occupied by
neovascular growth was counted, yielding a semiquantified measure of
severity with values ranging from 0 (no pathology) to 12 (most severe).
In this context, neovascular growth included any preretinal vascular
growth and any regions of dense arterial budding within the plane of
the superficial vessel plexus (see Fig. 6
, bottom center panel, open
arrow and black arrow). Neither of these two features is observed
during normal vessel development in room air–raised rats, and each is
seen in human ROP. Determinations were confirmed at 400x
magnification. Masked assessments were conducted by three independent
observers; the median of these individual assessments is reported. The
clock hour data from the present study were not normally distributed,
as determined by the D’Agostino test. Therefore, significant
differences were determined by the Kruskal–Wallis test with a
Scheffé’s post hoc analysis.
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Antisense RNA probe was generated from the 3'end of rat PAI-1 cDNA (a generous gift from T. D. Gelehrter, Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI). The SK(-) plasmid containing the PAI-1 cDNA was linearized by restriction digestion with HindIII. A 445 nucleotide-long 32P-UTP labeled cRNA was transcribed from 1 µg of the linearized template using T3 RNA polymerase (Ambion). A 210 bp nucleotide ß-actin antisense probe also was transcribed from linearized pTRIPLEscript vector (Ambion) using T3 RNA polymerase and was used as an internal standard for RNA levels. A 50-fold reduction in the specific activity of the ß-actin probe was achieved by the addition of cold UTP. This was necessary to increase the sensitivity of the assay, owing to the excess levels of ß-actin relative to PAI-1 mRNA in cells. The riboprobes were size-fractionated on 5% denaturing polyacrylamide gels and full length probes were eluted out of gel slices.
Coprecipitation of 2–8 x 104 cpm each of PAI-1 and ß-actin riboprobes (gel-purified) was performed with 0.3 to 0.6 µg individual PolyA+ samples using 50 µg tRNA as a carrier. The RNA/riboprobe coprecipitates were resuspended in Hyb-speed hybridization buffer (Ambion). Immediately after denaturation at 95°C for 4 minutes, samples were hybridized at 68°C for at least 10 minutes and digested at 37°C for 30 minutes, with 10x volume of an RNase cocktail (RNaseT1/RNase A at 1:2 vol:vol ratio) diluted at 1:100 in digestion buffer. The RNases were inactivated and the protected probe (PAI-1 and ß-actin) fragments were precipitated by the addition of 2x volume of an inactivation/precipitation buffer (Ambion). The precipitates were resuspended in formamide loading buffer and separated on a 5% denaturing polyacrylamide gel containing 7 m/l urea. In vitro transcribed, 32P-UTP labeled 100 to 500 bp fragments generated from a Century marker template were used as molecular weight markers. Gels were transferred to 3-mm paper, wrapped in plastic wrap and exposed to X-ray film with intensifying screen at -80°C from overnight to a week to obtain desired signal intensity for autoradiography.
The PAI-1 probe (
445 nt) is complementary to only 376 nt of the
PAI-1 mRNA sequence and is expected to produce a protected fragment of
that length. The ß-actin probe (210 nt) is expected to produce a 129
nt-protected fragment when hybridized to rat mRNA. Each treatment RNA
sample was extracted using retinas pooled from three pups.
In the presence of excess probe, the intensity of the protected bands are directly proportional to the relative levels of the message. With the housekeeping ß-actin gene serving as an internal control, the PAI/ß-actin pixel density ratio reflects the differences in PAI-1 mRNA levels between treatment groups.
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Results |
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Analysis of rats raised in the VO environment also demonstrated no significant effect of the steroid on retinal vascular area at either injection time, although a trend toward inhibition of intraretinal vessel growth was evident (Fig. 3A ). This effect was observed as a slight retardation of the extension of the primary vasculature toward the retinal periphery. Vascular areas were 18.2 ± 2.4 mm2 for anecortave acetate vs. 21.6 ± 3.2 mm2 for vehicle in retinas injected at day 14 (P = 0.10). Retinas that were injected at day 16 yielded vascular areas of 17.8 ± 2.7 mm2 for anecortave acetate vs. 21.5 ± 2.7 mm2 for vehicle (P = 0.07).
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Non-injected eyes of oxygen-exposed rats demonstrated more retinal vascular development (25.5 ± 4.5 mm2 than either vehicle- (P = 0.04) or drug-injected eyes (P < 0.001) at Day 14. On Day 16 results were P = 0.03 when compared to vehicle and P < 0.001 when compared to drug-injected eyes.
There was no apparent effect of vehicle or anecortave acetate on the vascular area of contralateral eyes. Rats receiving vehicle injections in one eye exhibited vascular areas in contralateral, uninjected eyes that were not different from those of rats receiving no injection in either eye. Rats receiving anecortave acetate injections in one eye exhibited vascular areas in contralateral, vehicle-injected eyes that were not different from those of vehicle injected eyes of rats with no injection in contralateral eyes.
To assess retinal neovascularization, we used a semiquantitative methodology involving a clock-face scale of neovascular growth severity (see Methods). This method was chosen because, although not rigorously quantitative, it has a clear corollary in the clinical classification of ROP and is widely accepted by laboratories that employ ROP animal models.27 35 36 37 38 39 40 Furthermore, multiple transverse sections are impractical and, because of the asymmetry of the pathology, are prone to sampling errors. Finally, when these sampling errors were controlled by serially sectioning entire eyes (700–900 sections/eye) in a similar rat model, the number of preretinal nuclei was highly correlated (r2 = 0.95, P < 0.0001) with clock-hour measures.41
There was significantly less retinal neovascularization in anecortave acetate–injected VOE rats than in vehicle-injected VOE rats, regardless of injection time (Fig. 3B) . Median values were 2 clock hours, ranging from 0 to 4 (mean ± SD, 2.1 ± 1.2 clock hours), for anecortave acetate vs. 6 clock hours, ranging from 3 to 8 (mean ± SD, 5.8 ± 1.6 clock hours) for vehicle at the first injection time (P < 0.0001). Median values resulting from the second injection were 3 clock hours, ranging from 1 to 5 (mean ± SD, 3.0 ± 1.1 clock hours), for anecortave acetate versus 6 clock hours ranging from 3 to 10 (mean ± SD, 5.9 ± 1.6 clock hours), for vehicle at the second injection time (P < 0.002). Among drug-treated eyes, there was no significant difference in the severity of neovascularization between treatment times (P = 0.21).
Noninjected eyes of oxygen-exposed rats showed significantly more pathologic neovascularization (median and range: 8, 5 to 12; mean ± SD, 8.2 ± 1.7 clock hours) than either vehicle- (median and range: 6, 3 to 10; mean ± SD, 5.8 ± 1.6, P < 0.0001) or drug-injected (median and range: 2.5, 0 to 5; mean ± SD, 2.5 ± 1.3, P < 0.0001) eyes, when data from both injection times are combined.
No room air–raised rat demonstrated retinal neovascularization. Control groups demonstrated no effect of vehicle or drug on neovascularization in contralateral eyes. Table 1 lists the critical values and confidence intervals for combined and paired comparisons of neovascular retinal clock hours.
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Discussion |
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In 1967, Pandolfi45 described a restructuring of extracellular matrix that occurred at actively extending vessel tips and that involved secretion of a protease known as plasminogen activator. It is now known that two distinct classes of plasminogen activators exist: a urokinase-type (u-PA), which participates in extracellular matrix breakdown during endothelial cell migration,46 and a tissue-type (t-PA), important in thrombolysis.47 Established vessels secrete only the t-PA form,48 but studies using a guinea pig corneal neovascularization model demonstrated that endothelial cells in new vessel sprouts secrete u-PA exclusively.49
Studies by Ashino-Fuse and colleagues50 explored the mechanism by which novel angiostatic steroids, already in use clinically for mammary carcinomas, exerted their effect. These workers concluded that angiostatic steroids suppressed PA activity, either by inhibiting production of the enzyme itself at the transcription or translation stage, or by inhibition of its secretion. The authors did not exclude the possibility of an increase in the level of an endogenous inhibitor of PA.
More recently, it has been demonstrated that angiostatic steroids exert their inhibitory effect on endothelial cell growth in vitro by increasing the synthesis of PAI-1.29 This induction of PAI-1 then inhibits u-PA activity, which is essential for the invasive aspect of angiogenesis — the breakdown of vascular endothelium basement membrane and extracellular matrix. Therefore, the result of steroid-induced suppression of PA function is that endothelial cells cannot proliferate and migrate toward an angiogenic stimulus to participate in new blood vessel formation. Our results provide the first evidence that angiostatic steroids may operate by the same mechanism in vivo. The anecortave acetate–mediated induction of PAI-1 mRNA is rapid (by 24 hours), is sustained (at least 3 days) and is robust (since constitutive levels of PAI-1 mRNA are undetectable by band densitometry in our system). The effect of this PAI-1 mRNA induction on downstream protease activity is currently under investigation.
Anecortave acetate is designed to be devoid of conventional hormonal activity. It contains the important structural modification of a 9–11 double bond that replaces the 11ß-hydroxyl group, which is essential for glucocorticoid and mineralocorticoid activities. Anecortave acetate demonstrates no significant glucocorticoid-mediated anti-inflammatory agonist activity in in vitro or in vivo inflammation assays, including carrageenan-induced footpad edema in rats, endotoxin-induced uveitis in rabbits, and IL-1 induction in cultured human U937 cells.15 51 In addition, anecortave acetate does not block the anti-inflammatory activity of dexamethasone, so it is also devoid of glucocorticoid antagonist activity.52 The absence of glucocorticoid activity is important because of the significant ocular side effects associated with ocular glucocorticoid therapy.
Trials of the angiostatic capacity of anecortave acetate have been conducted in chick chorioallantoic membrane15 ; in a rabbit corneal neovascularization model, where 90% inhibition of the area of new corneal blood vessels resulted18 ; and in an intraocular tumor model, where net tumor weight was held to less than 1/3 that of control.20 Tumor inhibition was concluded to be the result of the angiostatic properties of anecortave acetate, since neither the parent compound nor its deacetylated metabolite affected tumor cell proliferation in vitro. Notably, in each of the latter two studies the mode of administration was topical ocular application. Anecortave acetate has the additional attributes that it is relatively nontoxic (no studies describing its use have reported attrition), and its bioactivity is apparently independent of species or cause of the angiogenesis, making its therapeutic value more promising.
The present study demonstrates a phenomenon that our laboratory has observed over the past several years while delivering antiangiogenic agents intravitreally — namely, the therapeutic effect of vehicle injection. Preliminary studies have yielded an identical effect from dry needle puncture.53 In the present study, vehicle injections caused the reduction of retinal vascular area by approximately 15% and, more importantly, abnormal angiogenesis by approximately 30% when data from both injection times are combined. We postulate that the release of endogenous factors from the wound site and/or the surrounding retinal area may play a role in this effect, suggesting that other potential therapeutic agents for proliferative retinal disease might be constituents of the retina’s endogenous battery of cytokines. Based on this hypothesis, studies are underway to determine what endogenous retinal cytokines hold the capacity for antiangiogenesis and which one(s) are responsible for the effect of vehicle injection. In fact, the effect may be partly PAI-1–mediated, as indicated by the retinal response to vehicle injection illustrated in the pixel density bar graph (6B). Unfortunately, the single assay most representative of the effect of AA on PAI-1 induction in oxygen-treated rats (6A) did not show this vehicle effect.
ROP is a condition of growing concern in the United States. The incidence of blindness associated with this condition can be extrapolated from previous estimates21 to nearly 700 infants per year. Some permanent vision loss can be expected in nearly 4000 infants annually due to ROP. Among pathologic ocular conditions, ROP has the unique feature that normal and abnormal vessel growth occur simultaneously in very close proximity. The inhibitory effect of angiostatic steroids on normal retinal vascular development must be considered carefully before these, or like agents, can be developed for therapeutic application to ROP.
The limited effect of anecortave acetate on normal vessel development
(15% inhibition versus vehicle at either injection time), while it
profoundly affected pathologic neovascularization (50% or greater
inhibition versus vehicle, depending on injection time), is compelling.
At least two possibilities exist for this discrimination: 1) Early
intraretinal vessel development depends largely on differentiation of
mesenchymal precursors (vasculogenesis) rather than mitosis and budding
of existing vessels (angiogenesis), which produces the
neovascularization of ROP.54
It may be that basement
membrane and extracellular matrix remodeling, a known requirement of
angiogenesis,9
is less important to, or not required of,
retinal vasculogenesis. Thus inhibition of the proteases that perform
this remodeling function might preferentially target preretinal vessels
that grow by an angiogenic process. 2) Preretinal vessels may have been
available to the drug because of their immediate contact with the
vitreous, yet intraretinal vessels were not available. The ocular
pharmacokinetics of angiostatic steroids are only partly
defined,15
51
but daily examination of the drug-injected
eyes in this study would indicate that the highly hydrophobic
anecortave acetate was not rapidly cleared from the vitreous. In either
case, one must evaluate these drugs on the backdrop of the
invasiveness55
56
and limited favorable
outcome24
25
57
of cryo- or laser therapy — the currently
accepted methods of clinical intervention for ROP. When viewed in this
light, anecortave acetate or similar molecules may offer rational
alternatives to the current methods of surgical intervention.
Furthermore, in other retinopathies, where the retinal environment is
not complicated by normally developing vessels, this agent may inhibit
abnormal retinal angiogenesis while not effecting normal, mature blood
vessels in the same region.
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Acknowledgements |
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Footnotes |
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Submitted for publication April 24, 2000; revised August 16, 2000; accepted September 15, 2000.
Commercial relationships policy: E (RJC, AFC); N (all others). RJC and AFC are employees of Alcon Laboratories, which has a proprietary interest in anecortave acetate.
Corresponding author: John S. Penn, Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, 8016 Medical Center East, 2115 21st Avenue South, Nashville, TN 37232-8808. john.penn{at}mcmail.vanderbilt.edu
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