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(Investigative Ophthalmology and Visual Science. 2003;44:5423-5429.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0804
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Inhibition of Retinal Neovascularization by Intravitreal Injection of Human rPAI-1 in a Rat Model of Retinopathy of Prematurity

John S. Penn and Veera S. Rajaratnam

From the Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, Nashville, Tennessee.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE Restructuring of extracellular matrix at actively extending blood vessel tips involves secretion of plasminogen activator (PA). Findings in earlier studies conducted in the authors’ laboratory have suggested that angiostatic steroids suppress the PA activity essential for the invasive aspect of angiogenesis by increasing synthesis of plasminogen activator inhibitor (PAI)-1. This experiment was designed to test the effect of administration of exogenous PAI-1 on retinal neovascularization (NV) in an animal model of retinopathy of prematurity (ROP).

METHODS At birth, Sprague-Dawley rats were placed into incubators and exposed to an atmosphere alternating between 50% and 10% O2 every 24 hours. After 14 days, the animals were removed to room air, at which time each received a single intravitreal injection of 5 µL of buffer vehicle or one of five doses of PAI-1, ranging from 3.0 µg/mL to 2.0 mg/mL. Animals were killed 6 days later, and retinal NV was assessed using adenosine diphosphatase (ADPase) histochemical staining.

RESULTS Retinal neovascularization decreased with increasing PAI-1 dosage. The most effective dose tested (2.0 mg/mL) caused a 52% reduction in retinal NV relative to vehicle (P < 0.005). Normal vasculogenesis, as determined by measuring retinal vascular area, was unaffected.

CONCLUSIONS PAI-1 inhibits pathologic angiogenesis without adversely affecting normal vasculogenesis, an attractive feature for ROP therapies. Moreover, PAI’s relationship to matrix metalloproteinases, which are also implicated in angiogenesis, suggests that the proteolytic aspect of the process may provide additional downstream therapeutic targets.

Collectively, ocular conditions characterized by abnormal or poorly regulated vascular growth constitute the leading cause of blindness in the United States.1 2 This vascular growth occurs through the process of angiogenesis, wherein new vessels form from existing venules or venous capillaries after a set program that is apparently independent of the source of the initiating stimulus.3 This process is distinct from vasculogenesis, the formation of vessels by differentiation of pluripotent mesodermal precursor cells during fetal organ development.

The restructuring of extracellular matrix (ECM) at actively extending vessel tips by secretion of proteolytic enzymes is one of the well-characterized steps of the angiogenic program.4 These proteases generally belong to one of two classes: matrix metalloproteinases (MMPs) or serine proteases. Plasminogen activator (PA), a particularly important serine protease, exists in two forms: urokinase (uPA) and tissue (tPA). uPA participates primarily in ECM breakdown during endothelial cell migration,5 whereas tPA is important in thrombolysis.6 uPA cleaves a single bond in plasminogen, an inactive serine protease precursor, to yield the active protease plasmin, which has a broad specificity and cleaves a variety of proteins, including several important ECM components. Plasmin can also activate a battery of MMPs that contribute to ECM degradation.7

ECM remodeling during tumor growth and metastasis is a process that shares many features with angiogenesis. The interactions of promoters and inhibitors in this context have been reviewed extensively.8 9 Under normal conditions, proteases are kept under tight local control by protease inhibitors. MMPs are regulated by tissue inhibitors of metalloproteinases (TIMPs), whereas serine proteases are kept in check by serine protease inhibitors (serpins). Because TIMPs and serpins may play an important role in controlling angiogenesis, they have potential value for treating sight-threatening and other diseases characterized by angiogenic vessel growth or neovascularization.

In a previous study, the angiostatic steroid, anecortave acetate (AA; Alcon Laboratories, Inc., Fort Worth, TX), strongly inhibited abnormal retinal neovascularization (a product of angiogenesis), while only slightly affecting normal retinal vascular development (requiring both angiogenesis and vasculogenesis) in a rat model of retinopathy of prematurity.10 Steroids such as AA induce synthesis of plasminogen activator inhibitor (PAI)-1, thereby suppressing PA activity and preventing the breakdown of basement membrane and ECM and the consequent migration of endothelial cells to participate in the formation of new blood vessels.10 11 Human PAI-1 is a 42-kDa glycoprotein whose 379-amino-acid sequence, as deduced from its cDNA sequence, is homologous to members of the serpin superfamily of protease inhibitors.12 PAI-1 is unique in its ability to inhibit effectively both uPA and the single- and double-chain forms of tPA.13 The second-order rate constant describing the interaction of PAI-1 with uPA is on the order of 107 M-1, which is among the highest reported for any enzyme-inhibitor interaction.14

The experiments described in this work were designed to test the effect of intravitreal administration of human recombinant PAI-1 on retinal neovascularization in a rat model of retinopathy of prematurity. We chose this model because it affords several important advantages, which are detailed in the literature (e.g., Refs. 15 16 ).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Oxygen Exposure
Timed-pregnant Sprague-Dawley rats were delivered to the laboratory at approximately 18 days’ gestation. Within 4 hours after birth, the litter and mother were placed in an infant incubator (Isolette; Air Shields-Vickers, Batesville, IN), and the environment within the incubator was adjusted to 50% oxygen. After 24 hours, the oxygen concentration was rapidly reduced to 10%, where it remained for 24 hours. The oxygen continued to cycle between 50% and 10% every 24 hours for 14 days. The animals were then removed from the incubator to room air (21% oxygen). Intravitreal injections were given immediately on removal from the incubator (designated 14/0) for a dose–response trial. After preliminary time course experiments (n = 15), 2.5 days after exposure (hereafter designated 14/2.5) was chosen as the appropriate drug administration time for a more extensive trial at the most effective dose. All animals were killed after 6 days in room air (14/6), the time of peak retinal neovascularization in this model.17 18 Retinas were then dissected and stained by adenosine diphosphatase (ADPase) histochemistry for assessment of intraretinal vascular development and preretinal angiogenic vessel growth. Control rats were simultaneously raised in room air, treated, and killed. All experiments conformed to the published guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Treatment Groups
Human recombinant PAI-1 was obtained from DuPont Merck Pharmaceuticals (generous gift of Elizabeth Hausner, DVM, PhD). For the PAI-1 dose–response curve, concentrations of PAI-1 ranged from 3.0 µg/mL to 2.0 mg/mL (total n = 27). For the extended experimental trial, eyes were randomly assigned to the following treatment groups: no injection (n = 13), vehicle (10 mM phosphate-buffered saline; n = 13), PAI-1 (2.0 mg/mL; 47 µM; n = 21), tPA (3.0 mg/mL; 43 µM; n = 15), and PAI-1/tPA in combination (1:1 molar ratio, based on stoichiometry; n = 15). tPA was used rather than uPA, because its human recombinant form was commercially available (Activase; Genentech, Inc., San Francisco, CA).

Intravitreal Injections
Rats were anesthetized with methoxyflurane vapors (Metofane; no longer manufactured) and local application of 5% proparacaine. The eyelids were separated and the eyes mildly proptosed. With guidance from an operating microscope (Carl Zeiss Meditec, Inc., Thornwood, NY), a customized 30-gauge needle (Hamilton Co., Reno, NV), coupled to a 10-µL volume syringe (Hamilton), was inserted into the eye near the lateral canthus. After penetration of the globe approximately 0.5 mm posterior to the ora, 5 µL of the appropriate material was delivered to the posterior vitreous at the optic nerve head. Ophthalmic antibiotics (Alcon Laboratories, Inc.) were administered after injection to decrease the chance of infection.

uPA Activity
Room air–raised rats at P7 (n = 6) and oxygen-raised rats 2.5 days after exposure (n = 5) were injected with vehicle or PAI-1. One day later, the rats were killed, the eyes enucleated, and the retinas dissected and homogenized in ice-cold Tris buffer (pH 7.4). Aliquots containing sample were combined with assay buffer (uPA Activity Assay Kit; Chemicon International, Inc., Temecula, CA) in a 96-well plate according to the manufacturer’s instructions. After a 12-hour incubation of this mixture with chromogenic substrate (1:1 by volume; Chemicon International, Inc.) at 37°C, absorbance was read at 405 nm (SpectroMax 190; Molecular Devices, Sunnyvale, CA). Absorbances were converted to units of activity using standard curves generated with pure uPA enzyme provided with the kit. Activity is expressed per milligram of total sample protein, as determined by a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL).

Gel Zymography
Other rats were injected with 2.0 mg/mL or 0.2 mg/mL rPAI-1 or vehicle at 14/2.5 and were killed 24 hours later. After retinal dissection, two retinas from each treatment were pooled and homogenized in 150 µL of extraction buffer (40 mM Tris-HCl, 110 mM Tris base (pH. 7.4), 150 mM NaCl, 5 mM CaCl, 5 mM MgCl2, and 1% Triton X-100) before flash-freezing. Samples were thawed and centrifuged at 20,800g for 8 minutes at 4°C. Protein concentration was measured in all samples with the BCA protein assay kit (Pierce), and an equivalent volume of each was affinity purified with an 8:1 ratio of sample volume to gelatin Sepharose 4B beads (Amersham Pharmacia Biotech, Piscataway, NJ) by incubation at 4°C with rocking for 1 hour. Samples were eluted in 30 µL of 2x Bio-rad zymogram sample buffer (Richmond, CA) plus 10% dimethyl sulfoxide (DMSO). A 20-µL aliquot of each sample was loaded on a 10-well, 10% gelatin zymography gel (Ready Gel; Bio-Rad) with appropriate markers and controls. The gel was run for 90 minutes at 100 V in 1x Tris-glycine-SDS buffer (20 mM Tris base, 200 mM glycine, 3 mM SDS). After incubation with shaking at 25°C in 1x zymogram renaturation buffer (Bio-Rad; 2.5% Triton X-100) for 45 minutes, the gel was left overnight (16–20 hours, optimally) at 37°C in 1x zymogram development buffer (50 mM Tris-HCl [pH 7.5], 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35; Bio-Rad). The gel was stained for 20 minutes in Coomassie blue stain (0.5% Coomassie blue R-250, 40% methanol, 10% acetic acid in distilled water), rinsed briefly in distilled water, and then destained for up to 2 hours in 40% methanol plus 10% acetic acid. Zones of clearing that corresponded to the presence of proteinases in the gel were quantified using image-analysis software (Enhance; MicroFrontier, Des Moines, IA). The data are expressed as pixels per microgram protein.

In Situ Zymography
Five days after removal from the exposure chamber, some rats were administered 2.0 mg/mL rPAI-1 by intravitreal injection and killed 24 hours later. The timing of injection and death were chosen to ensure that there were already preretinal neovascular tufts at the time the agent was administered. Eyes were enucleated, cryoprotected without fixation by infiltration in 5% and then 30% sucrose, infiltrated with optimal cutting temperature (OCT) cryoembedding compound (Tissue-Tek; Sakura Finetek, USA, Inc.; Torrance, CA), and frozen at -20°C. Microscope slides were evenly coated with 40 µL of substrate containing fluorescein-conjugated gelatin (DQ-Gelatin; Molecular Probes, Eugene, OR) in 2% gelatin, 2% sucrose, and 0.2% sodium azide in PBS. The slides were stored overnight at 4°C and protected from light. The following day, 8-µm-thick frozen tissue sections were placed directly onto the slides where they incubated under humid conditions for 16 hours at 42°C. Slides were viewed using appropriate fluorescence filters and photographed at 100x or 200x magnification (AX70 Provis Photomicroscope; Olympus Optical Co., Tokyo, Japan, with Tmax 100 film; Eastman Kodak, Rochester, NY). Navigation of the sections was facilitated by subsequent staining with 0.1 µg/mL 4',6-diamidino-2-phenolindole, dihydrochloride (DAPI; Molecular Probes) in Tris-buffered saline (TBS) for 10 minutes, followed by washing in TBS.

Retinal Angiogenesis
To determine the effect of the various treatments on angiogenesis, the extent of retinal neovascularization was assessed in flattened retinas stained for ADPase activity.19 Images of ADPase-stained retinas were digitized, captured, and displayed at 65x magnification. Vessel tufts were then outlined directly on the monitor face with an interactive stylus pen (FTG Data Systems, Stanton, CA). The pixels contained within an encircled area were counted, the total number of pixels from all areas was summed, and this value was converted to square millimeters. Where there was a question of the preretinal nature of the tuft, the tissue was evaluated simultaneously with a microscope at 200x magnification, using the plane of focus. This method of estimation correlates well (r2 = 0.947) with the clock-hour method of estimation used by us and others,20 21 22 23 24 and it is much less time consuming than preretinal cell counts in multiple transverse sections. In addition, it eliminates the sampling bias inherent in choosing which transverse retinal sections are used for counting nuclei and which retinal regions are included in the counted sections. Moreover, unlike clock-hour measures, this method yields normally distributed data that allow statistically significant differences between treatment groups to be determined by analysis of variance. A Bonferroni-Dunn post hoc procedure was used to determine differences.

Vascular Area
To determine the effect of the various treatments on vasculogenesis, the extent of vascular growth associated with normal retinal development was measured in 7- and 20-day-old rats. Images of ADPase-stained retinas were digitized, captured, and displayed at 20x magnification. The retinal area containing blood vessels was traced on the monitor face with a stylus pen,10 and the number of pixels within this area was converted to square millimeters. Measurements of this parameter were recorded, and statistically significant differences between the treatment groups were determined by analysis of variance with a Bonferroni-Dunn post hoc procedure.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The semilogarithmic plot of PAI-1 dose versus area of retinal neovascularization (Fig. 1A) was reasonably linear (r2 = 0.883), and its slope was significantly different from zero (P < 0.01). Conversely, there was no effect of increasing PAI-1 concentration on retinal vascular area (slope not significantly different from zero; Fig. 1B ). Based on this preliminary information, a concentration of 2.0 mg/mL (47 µM) was chosen for extended trials with appropriate control groups. Because the injection volume of 5 µL is diluted in approximately 25 µL of vitreous, the effective vitreous concentration was approximately 8 µM.



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  		FIGURE 1. A dose–response curve (mean ± SD) describes the effect of human rPAI-1 on pathologic angiogenesis (A) 6 days after removal from the exposure chamber and on normal retinal vessel growth (B) at the same time. PAI-1 was administered immediately after removal of rats from the exposure chamber. In order of increasing dose, sample sizes were five, five, six, six, and five for the separate treatment groups.

 
Injections given at 0, 1, 2, 2.5, or 3 days after exposure resulted in no statistically significant differences in disease outcome between times, although each of these injections performed significantly better than those given at 4 or 5 days after exposure. Subsequent injections were given at 2.5 days after exposure because this injection time resulted in a slightly greater reduction in retinal neovascularization than any of the other early after-exposure time points (data not shown).

As shown in Table 1 and Figure 2A , vehicle-injection at 2.5 days after exposure resulted in significantly less retinal neovascularization than no injection at day 6 after exposure (2.35 ± 0.22 vs. 3.02 ± 0.14 mm2; P = 0.0427). PAI-1–injected eyes showed significantly less retinal neovascularization than vehicle-injected eyes (1.12 ± 0.13 vs. 2.35 ± 0.22 mm2; P < 0.005), and tPA-injected eyes had significantly more retinal neovascularization than PAI-1-injected eyes (2.70 ± 0.21 vs. 1.12 ± 0.13 mm2; P < 0.001). PAI-1-injected eyes also showed less retinal neovascularization than PAI-1+tPA-injected eyes (1.12 ± 0.13 vs. 2.12 ± 0.23 mm2; P = 0.0043). One final notable comparison showed the predictable trend, although the difference is not significant: tPA-injected eyes showed more retinal neovascularization than PAI-1+tPA-injected eyes (2.70 ± 0.21 vs. 2.12 ± 0.23 mm2; P = 0.0576).


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  		TABLE 1. Critical Variables and Confidence Intervals for Comparisons of Neovascularization Measures

 


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  		FIGURE 2. At the optimal dose and time, human rPAI-1 significantly inhibited retinal neovascularization (A). When PAI-1 was combined with a molar equivalent of tPA before injection, its inhibitory effect was abrogated. Normal retinal vessel growth, as estimated by measurements of retinal vascular area, was not similarly effected (B). All agents were administered 2.5 days after removal of rats from the exposure chamber. Data are the mean ± SD.

 
Figure 2B depicts the effect of intravitreal injection of human rPAI-1 on retinal vascular area at 6 days after exposure. Analysis of variance yielded no significant difference in vascular area between any of the treatment groups.

Figure 3 includes representative retinas from three important treatment groups: room air–raised (A); oxygen-exposed, vehicle-injected (B); and oxygen-exposed, PAI-1–injected (C). There was no apparent effect of vehicle or any other agent or combination of agents on retinas from room air–raised rats.



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  		FIGURE 3. Images of representative retinas from three of the five main treatment groups: (A) a room air–raised rat receiving 47 µm PAI-1, (B) an oxygen-exposed rat receiving PBS vehicle, and (C) an oxygen-exposed rat receiving 47 µm PAI-1. Arrows: examples of preretinal neovascular tufts.

 
Retinal uPA activity was significantly inhibited by PAI-1 injection as shown in Figure 4 . In room air rats treated at P7, a time when normal vasculogenesis is ongoing, activity was reduced 26% in PAI-1–injected eyes, relative to vehicle (P < 0.05). There was no difference in retinal vascular area between these two groups when assessed at postnatal day (P)10. Oxygen-treated rats injected at 2.5 days after exposure showed a 75% inhibition of retinal uPA activity 1 day later (P < 0.001).



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  		FIGURE 4. In room air–raised rats, intravitreal injection of PAI-1 at P7 reduced retinal uPA activity by 26% (*P < 0.05) by P8, but resulted in no effect on retinal vascular area when measured at P10. In oxygen-treated rats injected with PAI-1 at P16.5 and assessed 1 day later, retinal uPA activity was reduced 75% relative to those of vehicle-treated rats (**P < 0.001).

 
Zymograms produced using retinal tissue from eyes injected 2.5 days after exposure and enucleated for retinal dis1 day later showed a dose-dependent relationship between PAI-1 and MMP-2 and -9 levels. Table 2 summarizes the effect of vehicle and PAI-1 on the latent and activated forms of these two metalloproteinases. Activated MMP-2 was reduced nearly threefold in eyes injected with 2.0 mg/mL PAI-1 relative to injection of vehicle. Activated MMP-9 was decreased similarly by the same treatment.


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  		TABLE 2. Zymographic Analysis of MMP-2 and -9

 
Finally, as illustrated in Figure 5 , tissue zymography revealed reduced protease activity associated with neovascular tufts in PAI-1–injected eyes compared with vehicle-injected eyes. In age-matched room air–raised rats, the technique demonstrated incomplete retinal penetration of the effect of PAI-1 (Fig. 5E) . Activity in the superficial vessels was completely absent, whereas the deep retinal vessels displayed normal or nearly normal activity. This is reasonably consistent with the results of the uPA assay, although the two were conducted in animals of slightly different age (P16.5 vs. P20).



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  		FIGURE 5. In situ zymography demonstrated decreased gelatinase activity in transverse retinal cryosections from eyes receiving vehicle injection at 14/5 and undergoing assessment 1 day later. (A), relative to PAI-1 injection at the same time (B). The same sections are stained with DAPI after zymography to facilitate visualization of the preretinal vessel tufts in vehicle- and PAI-1-injected retinas (C, D, respectively). (E) Retina from a room air–raised control rat injected with PAI-1 on P19 and killed for tissue preparation 1 day later. Gelatinase activity associated with superficial retinal vessels appeared completely inhibited, whereas the deep retinal vessels retained activity. Arrows: position of gelatinase-active vessels within the deep retinal capillary net. ILM, inner limiting membrane; INL, inner nuclear layer; ONL, outer nuclear layer; ROS, rod outer segments. Magnification bar: (A–D) 20 µm; (E) 40 µm.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Intravitreal injection of human recombinant PAI-1 significantly reduced retinal neovascularization in a rat model of ROP. The highest level of inhibition observed was 52%, which compares favorably with the performance of other angiostatic compounds in this model (e.g., Refs. 10 18 ), but also strongly supports the role of other pathways unrelated to proteolysis but crucial to the angiogenesis process. tPA negated the action of PAI-1 when the two compounds were administered simultaneously. Conversely, none of the various compounds used (PAI-1, tPA, or PAI-1+tPA) had a significant effect on retinal vascular area, suggesting that the vasculogenic component of retinal blood vessel formation was not similarly inhibited. There are two possible explanations for this discrimination: Either the pathologic vessels were specifically targeted, or the PAI-1 did not reach the normal intraretinal vessels. Initial growth of the intraretinal vasculature is largely a process of cellular differentiation (vasculogenesis), which may not require induction of proteolysis and ECM breakdown, whereas later growth and remodeling almost certainly require these events. Unfortunately, our assessment of vascular area is probably too crude to have identified alterations of vessel remodeling by PAI-1 treatment, although injection of room air–raised rats of two ages resulted in no apparent alteration of retinal vessel density or architecture (Fig. 3A) . Notably, transgenic mice that overexpress PAI-1 also show development of a relatively normal retinal vasculature, indicating that this process is not strongly influenced by excessive enzyme.25 It is also possible that the agents used did not effectively penetrate the retina, rendering them bioavailable to preretinal, but not all intraretinal, sites of inhibition. However, the molecular weights of PAI-1 and tPA argue for effective retinal penetration,26 and the in situ and gel zymograms and the uPA activity assay all support the notion that at least partial retinal penetration and inhibition occur. Discrimination between preretinal and intraretinal vessels by PAI-1 or any other therapy is particularly attractive in the context of treating ROP, because normal intraretinal vascular growth and pathologic preretinal vessel growth occur simultaneously in close proximity.

Zymographic analysis of retinal tissue provided evidence consistent with two previous studies that have pointed to the role of protease activity in ocular angiogenesis.27 28 Our analysis, like those of others, focused on MMP-2 and -9, because they have specificity for the substrates composing the subendothelial matrix.28 29 In the present study, injection of 2.0 mg/mL PAI-1 resulted in a 63% reduction in the activated form of MMP-2, relative to vehicle injection, whereas the activated form of MMP-9 was reduced by 56%. MMP-9 apparently was strongly induced by the injection (Penn JS, et al. IOVS 2000;41:ARVO Abstract S142; Rajaratnam VS, et al. IOVS 2001;44:ARVO Abstract 513), because even the vehicle injection resulted in a 2.5-fold increase in latent and activated forms, combined. The result of this induction is that PAI-1–treated eyes, which showed the lowest degree of neovascularization, had substantially more latent and activated MMP-9 than noninjected eyes, which had the most disease. This suggests that MMP-9 may not have been a crucial component of the angiogenic process in this model system. Conversely, the level of activated MMP-2 in retinas of PAI-1 injected eyes was only one fourth that of noninjected eyes, indicating that it may have provided an important inhibitory influence. The notion that MMP-2 plays a more prominent role in angiogenesis than MMP-9 is not new,30 31 32 33 34 but the relative contributions of these two enzymes to retinal angiogenesis probably will not be determined until specific inhibitors are developed and used.

The active form of PAI-1 has been shown to convert spontaneously to a latent inactive form at 37°C.35 However, PAI-1 is maintained in an active form, and its inhibitory activity is preserved when it is bound to vitronectin in subendothelial matrix.36 Vitronectin can affect angiogenesis through its interaction with endothelial cell integrins, particularly {alpha}vß3 and {alpha}vß5.37 Peptide antagonists containing the RGD sequence through which this interaction occurs have been shown to inhibit angiogenesis in vivo.38 More relevant to the present study, a PAI-1 mutant protein that binds vitronectin, but does not inhibit plasminogen activation, was successful in inhibiting migration of human WISH cells and human epidermoid carcinoma Hep-2 cells.39 Thus, PAI-1 may act to influence angiogenesis independent of its role as a protease inhibitor by interfering with endothelial cell binding to vitronectin within the matrix.39 40

Our use of tPA as a control treatment was a preliminary attempt to discriminate the relative inhibition of matrix dissolution and cell migration and attachment by human rPAI-1 in our model. In addition, the results of the gel and in situ zymography argue that proteolysis was indeed inhibited in rats receiving PAI-1. Although neither finding is conclusive, they suggest that, in the context of rat ROP, ECM digestion is a critical component of retinal neovascularization. Hence, the data advocate protease inhibition as a reasonable therapeutic strategy for conditions in which retinal angiogenesis plays a critical role. Still, antiangiogenesis and antiproteolysis are not necessarily correlated.41 A reduction in extracellular proteolysis is expected to reduce the ability of endothelial cells to overcome the mechanical barriers imposed by the surrounding ECM. However, because proteases can also modulate cytokine activity, for example by liberating matrix-bound angiogenic growth factors like vascular endothelial growth factor,42 43 the relationship between matrix proteolysis and angiogenesis is likely to be varied and complex. We plan to investigate this issue in our model by using specific MMP inhibitors that have no demonstrated effect on the PA system or on endothelial cell attachment and migration. One such class of inhibitors comprises the zinc chelators.

This caveat notwithstanding, evidence is accumulating to support the therapeutic potential of synthetic and natural PA inhibitors in ocular and other sites of angiogenesis. Presently, the predominant clinical treatment for neovascular retinopathies, ROP included, is laser photocoagulation. This treatment has proven value, but it does not always prevent the vasoproliferative condition from recurring, and it can have adverse effects, such as reduced peripheral and night vision. Other avenues of therapy for retinal angiogenesis have thus been sought, and the initial focus has been on vascular endothelial growth factor, its endothelial cell surface receptors, and the transduction cascade initiated by them.44 45 46 47 48 Herein, we have extended the focus to another early and common step in the angiogenesis process—endothelial cell digestion of ECM.

The use of more specific (downstream) inhibitors of protease activity may show yet greater promise, particularly if the specific matrix metalloproteinases involved can be targeted. Additional therapeutic value may be achieved by using protease inhibitors such as PAI-1 or MMP inhibitors in combination with agents targeting other aspects of angiogenesis, such as growth factor induction, cell signaling or cell attachment, and migration. Combination antiangiogenic therapies would mimic the strategy used in cancer treatments, wherein combinations of chemotherapeutic drugs are often used. In this way, the antiangiogenic potential can be maximized, while the toxic side effects are minimized. Collateral toxicity has particular significance in the retina, which is populated with fragile, postmitotic neurons. Because PAI-1 is endogenous to the retina, the possibility of altering its synthesis and release has distinct advantages over administration of exogenous, and potentially toxic, synthetic antiangiogenic drugs.


   Footnotes
 
Supported in part by National Eye Institute EY07533 (JSP), a Wasserman Merit Award to JSP from Research to Prevent Blindness (RPB), Inc., and a Challenge Grant to Vanderbilt Ophthalmology from RPB.

Submitted for publication August 8, 2002; revised May 1, 2003; accepted July 14, 2003.

Disclosure: J.S. Penn, DuPont Merck Pharmaceuticals (F); V.S. Rajaratnam, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: John S. Penn, Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, 2115 21st Avenue South, 8016 Medical Center East, Nashville, TN 37232-8808; john.penn{at}vanderbilt.edu.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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