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Laboratory Sciences |

Adenovirus-Mediated Gene Therapy Using Human p21WAF-1/Cip-1to Prevent Wound Healing in a Rabbit Model of Glaucoma Filtration Surgery FREE

Todd W. Perkins, MD; Barbara Faha, PhD; Ming Ni, MD; Julie A. Kiland; Gretchen L. Poulsen; Doug Antelman, PhD; Isabella Atencio, PhD; Jeremy Shinoda; Dinesh Sinha, PhD; Lyndia Brumback, MS; Daniel Maneval, PhD; Paul L. Kaufman, MD; Robert W. Nickells, PhD
[+] Author Affiliations

From the Departments of Ophthalmology and Visual Sciences (Drs Perkins, Kaufman, and Nickells and Mss Kiland and Poulsen) and Biostatistics and Medical Informatics (Ms Brumback), University of Wisconsin, Madison; Canji, Inc, San Diego, Calif (Drs Faha, Ni, Antelman, Atencio, and Maneval and Mr Shinoda); and Schering Plough Research Institute, Lafayette, NJ (Dr Sinha). Drs Kaufman and Nickells have worked as consultants to Canji, Inc.


Arch Ophthalmol. 2002;120(7):941-949. doi:10.1001/archopht.120.7.941.
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Objective  To determine if adenovirus-mediated p21WAF-1/Cip-1 (p21) gene therapy can prevent fibroproliferation and wound healing in a rabbit model of glaucoma filtration surgery.

Methods  In vitro studies were performed using rabbit Tenon fibroblasts harvested from fresh tissue. In vivo studies were conducted in New Zealand white rabbits. A full-thickness sclerotomy was performed under a limbal-based conjunctival flap. Reagents tested included a replication-deficient recombinant adenovirus containing the human p21 gene (rAd.p21); the nonspecific marker gene for green fluorescent protein or β-galactosidase; mitomycin, 0.5 mg/mL; and balanced saline solution. Each treatment was applied episclerally for 5 minutes before the sclerotomy using a soaked cellulose sponge placed under the surgically created conjunctival flap. Independent experiments were conducted to (1) monitor changes in intraocular pressure during a 30-day period after treatment and examine surgical site histological features, (2) examine changes in bleb morphologic features over 30 days, (3) determine outflow facility 14 days after treatment, and (4) examine the localization and persistence of rAd.p21 expression between 3 and 60 days after treatment.

Results  Treatment of Tenon fibroblasts with rAd.p21 resulted in a dose-dependent inhibition of DNA synthesis and cell growth in vitro. In vivo, rAd.p21 inhibited wound healing and fibroproliferation after filtration surgery, comparably to mitomycin. Mitomycin caused notable thinning of the bleb wall. In addition, 2 of the 5 mitomycin-treated eyes exhibited an abscess with hypopyon and hyalitis 30 days after surgery, which was not observed in any of the rAd.p21-treated eyes. None of the treatments resulted in a significantly sustained decrease in intraocular pressure during the 30-day period, although mitomycin treatment resulted in a significant (P = .02) increase in outflow facility 2 weeks after surgery in separate animals. Mitomycin- and rAd.p21-treated eyes had functioning blebs at the end of the experiment based on slitlamp examination.

Conclusions  Mitomycin and rAd.p21 were effective in preventing fibroproliferation and wound healing in a rabbit model of glaucoma surgery. Mitomycin treatment increased outflow facility in normal-pressure eyes.

Clinical Relevance  Gene therapy with rAd.p21 may provide an effective antiproliferative for glaucoma filtration surgery, without the complications associated with mitomycin.

Figures in this Article

CONTROL OF the mammalian cell cycle is a highly regulated process that requires the interactions of multiple gene products. Several gene products have been identified that act to prevent progression through the cell cycle. Two of these are the tumor suppressor protein p531 and its downstream effector protein p21WAF-1/Cip-1 (p21).24 The p21 protein negatively regulates the cell cycle by binding to and inhibiting the activity of cyclin-dependent kinases, a family of proteins required for the transition from the interphase(G1) to the S phase (the phase during which DNA is synthesized) of the cell cycle (Gartel et al5 provide a review). Inhibition of cyclin-dependent kinases results in cell cycle arrest at the G1-S interface, and several studies68 have shown that exogenous expression of p21 can inhibit cell proliferation.

Treatment of cancer has been a primary focus of gene therapy protocols and clinical trials using cell cycle regulatory genes such as p53.9,10 However, other medical conditions that result from nonmalignant cell proliferation are candidates for gene therapy with cell cycle regulators. One example is the fibroproliferation that occurs as part of the normal wound healing response after trabeculectomy for glaucoma, resulting in failure of the surgery. The use of antiproliferative agents, such as fluorouracil and mitomycin, has greatly increased the success of filtration surgery, but these agents are associated with complications ranging from corneal epithelial defects to late-onset bleb leaks that can lead to endophthalmitis.11 These complications have prompted the search for alternative reagents that provide the desired antiproliferative effect without the associated complications.1214 In this study, we examined the potential of gene therapy using human p21 to prevent wound healing in a rabbit model of filtration surgery. The results show that an adenovirus containing the human p21 gene (rAd.p21) can effectively block the proliferation of Tenon fibroblasts and inhibit scarring during a 30-day period in the rabbit eye, without some of the extreme effects found with mitomycin.

ANIMALS

New Zealand white rabbits (Pasteurella free) were used for this study. Experimental procedures were conducted according to the guidelines established by the Association for Research in Vision and Ophthalmology statement for the use of animals in research and approved by the Animal Use Committee at the University of Wisconsin, Madison. These animals were used to evaluate the effect of rAd.p21 in glaucoma filtration surgery in 3 independent series of experiments. A total of 20 rabbits were used to evaluate changes in intraocular pressure (IOP) and surgical site histological features, 18 were used to determine changes in bleb characteristics, and 15 were used to measure outflow facility. An additional 12 rabbits were used for separate experiments to determine the localization and persistence of rAd.p21 expression. All experimental analyses using rabbits were conducted in a masked fashion, including surgery, IOP measurements, bleb scoring, outflow facility measurements, and the determination of histological features.

RECOMBINANT ADENOVIRUS

An E1/partial E3–deleted recombinant adenovirus was used for gene transfer in these experiments. Essentially, the p21 coding region, driven by the human cytomegalovirus immediate early promoter, was cloned into the E1 region of the adenovirus (rAd.p21). Control viruses were created in a similar manner; they encode bacterial β-galactosidase, jellyfish green fluorescent protein, or no added transgene (rAd.control) in place of the p21 coding sequence. Deletion of the E1 region of adenovirus renders the virus replication deficient. Details of the construction of the recombinant adenovirus are published elsewhere.15 Recombinant viruses were grown and propagated in the human embryonic kidney cell line 293 (American Type Culture Collection, Rockville, Md) and purified using standard protocols.16

IN VITRO STUDIES

Tenon fibroblasts were harvested from explants of the Tenon capsule dissected from freshly enucleated rabbit eyes and grown in Dulbecco Modified Eagle Medium (DMEM), containing 10% fetal bovine serum (FBS); penicillin G sodium, 100 U/mL; streptomycin sulfate, 100 µg/mL; and amphotericin B, 0.25 µg/mL. Only cells before passage 10 were used for in vitro analyses. Before recombinant adenovirus treatment, cells were made quiescent by serum starvation (with 0.5% FBS) for 24 hours. For thymidine incorporation experiments, quiescent cells plated in 96-well plates were incubated overnight with rAd.p21 or rAd.control (in triplicate) with increasing virus particle concentrations(4 × 107-3 × 109 virus particles per milliliter). Cells were then washed and refed with complete media containing tritiated thymidine. After 24 hours, plates were harvested and incorporated tritiated thymidine was counted (TopCount Microplate Scintillation Counter; Packard BioScience Co, Meriden, Conn).

For cell growth assays, quiescent cells were incubated with rAd.p21 or rAd.control (6.7 × 107 virus particles per milliliter) for 24 hours, after which the recombinant adenovirus was removed by washing. The cells were stimulated to grow by the addition of complete media. Five days later, cell growth was evaluated by counting viable cells using trypan blue exclusion.

For cell cycle analyses, primary human ocular fibroblasts from the Tenon capsule obtained during surgery were isolated and cultured as previously described. Cells were arrested by culturing in DMEM plus 0.5% FBS for 3 days. Arrested cells were treated overnight with 1 × 108 virus particles per milliliter of rAd.p21 or rAd.control. Cells were washed in DMEM plus 10% FBS to remove the adenovirus, and released from G0 (the quiescent phase of the cell cycle) or G1 by culturing in DMEM plus 10% FBS for 24 hours, at which time they were treated with trypsin and fixed in 70% ethanol at 4°C overnight. Cells were washed with phosphate-buffered saline(PBS), centrifuged at 1500 rpm, and treated with RNase, 5 µg/mL, for 30 minutes at 37°C. Cells were stained with propidium iodide, 50 µg/mL(Molecular Probes, Inc, Eugene, Ore), for 30 minutes, followed by flow cytometric analysis. Computer software (Cell Quest; Becton, Dickinson and Company, Franklin Lakes, NJ) was used to quantify percentages of cells in each phase of the cell cycle.

SCLEROSTOMY PROTOCOL

Surgery was conducted on one eye of each rabbit for IOP and bleb evaluation studies. In some cases, both eyes of one rabbit were operated on in animals used for outflow facility measurements. The bilateral surgery protocol was approved by the Animal Use Committee at the University of Wisconsin, providing that one eye received balanced saline solution (BSS) only as the antiproliferative agent. Rabbits were anesthetized with an intramuscular injection of ketamine hydrochloride, 40 mg/kg body weight, and xylazine hydrochloride, 5 mg/kg body weight. A wire eyelid speculum was placed, and the eye was fixed with a corneal suture (6-0 nylon). A limbal-based flap of the conjunctiva and the Tenon capsule was made in the superior nasal quadrant of either left or right eyes, and the sclera was cauterized. Each reagent was soaked into a 4 × 4 × 1-mm cellulose sponge, which was placed on the sclera under the flap for 5 minutes. After application, the area was irrigated extensively with BSS. The irrigation step was omitted in some cases, with no effect on any of the variables examined in this study (such as the level of transduction of fibroblasts). For the sclerostomy, a limbal groove angled toward the anterior chamber was first made with a No. 57 blade, followed by the production of a fistula first with a No. 75 blade and then with a Kelly punch. About 2 to 3 punches were generally taken in each eye. An iridectomy was then performed through the fistula, and the conjunctiva was closed with a 9-0 polyglactin suture. After surgery, 1 cm each of 1% atropine sulfate ophthalmic ointment and neomycin sulfate, 3.5 mg/g; polymyxin B sulfate, 10 000 U; and dexamethasone, 1 mg/g, ointment was applied to the eye.

TRANSGENE EXPRESSION AND DISTRIBUTION OF VIRAL INFECTION

Immediately following euthanasia, eyes were enucleated and rinsed in PBS. The surgical site was dissected, placed in a cryovial, and snap frozen in liquid nitrogen. Total RNA was extracted from approximately 100 mg of tissue harvested from the surgical sites of 3 eyes, using a reagent (Tri-Reagent; Molecular Research Center, Inc, Cincinnati, Ohio), per the manufacturer's protocol. Total RNA was treated with DNase I (Roche/Boehringer-Mannheim, Berkeley, Calif) to remove residual DNA. Quantification of human p21 messenger RNA was performed using reverse transcription–polymerase chain reaction with real-time detection (PE Applied Biosystems, Foster City, Calif), as described previously.17 The primers used were as follows: forward primer, 5′-AACGGTACTCCGCCACC-3′; reverse primer, 5′-TTCTGACATGGCGCCTACT-3′; and probe, 5′-FAM-TCCGCATCGACCGGATCGG-TAMRA-3′. The protein from the remaining tissue homogenate was then extracted for the detection of p21 by enzyme-linked immunosorbent assay per the manufacturer's protocol (Oncogene Research Products, San Diego, Calif).

Expression of the p21 transgene in vivo was detected by immunohistochemistry using a monoclonal antibody against human p21 that exhibited no cross-reactivity to the rabbit homolog (Oncogene Sciences, Inc, Uniondale, NY). Briefly, globes from rabbits treated with each reagent were harvested at different times after surgery, fixed in 4% paraformaldehyde in phosphate buffer (pH, 7.2) for 48 hours at 4°C, and embedded in paraffin. Paraffin sections (5 µm) were cut and stained for p21 expression, as previously described.18

EVALUATION OF EFFICACY
IOP Measurements

Intraocular pressure was measured in awake rabbits before and after surgery using an applanation tonometer (Tono-Pen XL; Mentor Corp, Norwell, Mass).19 All IOPs were obtained at the same time each day by one of us (J.A.K.). Baseline IOPs were determined from average measurements taken one to several days before surgery after the rabbits became familiar with their handler. Intraocular pressure was monitored every 2 to 3 days after surgery for the first 10 days and every 3 to 4 days thereafter for the remaining 20 days.

Bleb Evaluation

Blebs were evaluated during slitlamp examinations on a qualitative scale of 1+ to 4+, reflecting increasing bleb height and size as follows: 1+, minimal height, conjunctiva thickening, and no microcysts; 2+, microcysts are present; 3+, elevated bleb covering 3 to 4 clock hours of the eye; and 4+, greatly elevated bleb covering longer than 5 clock hours of the eye. A score of 0 indicates no observable bleb. Controls for these experiments were conducted using sponges soaked in PBS containing 3% sucrose, which is a solution used to maintain viral stability during cryostorage.

Histological Evaluation

For this study, the rabbits were killed humanely 30 days after surgery. A small burn was made with an ophthalmic cautery in the cornea of each eye to mark the surgical area before enucleation. An incision was made 90° away from the surgical site, and the whole globe was fixed in 4% paraformaldehyde in phosphate buffer (pH, 7.2) for 48 hours at 4°C and embedded in paraffin. Serial sections were then cut through the sclerostomy site. Approximately every fifth section was stained using Gomori trichrome.20 Sections were masked and evaluated by 2 pathologists who scored the extent of fibroproliferation and cellular infiltrate using the following scale: 0, no change; 1, minimal change; 2, mild change; 3, moderate change; and 4, severe change. A final score for each variable was made by consensus between the 2 observers.

Outflow Facility

Total outflow facility was determined using 2-level constant-pressure anterior chamber perfusion with Bárány21 solution. Animals were evaluated 14 days after surgery, after the time when sclerostomies performed with no antifibrotic agent typically fail. Generally, each eye was perfused for 60 minutes, during which 9 outflow facility measurements were taken. The calculated outflow facility for each eye was then taken as the average of these measurements.

IN VITRO STUDIES

Treatment of quiescent rabbit Tenon fibroblasts with rAd.p21 resulted in a dose-dependent inhibition of DNA synthesis within 24 hours after infection, as measured by tritiated thymidine incorporation (Figure 1A). No inhibition was observed with control treatments. Fluorescent-activated cell sorter analysis of similarly treated human Tenon fibroblasts showed that inhibition of DNA synthesis by rAd.p21, but not by rAd.control, resulted in a G1 arrest of the cell cycle (Table 1). The rAd.p21-treated cells were viable, as there was no sub-G1 population indicative of debris from dead cells (not shown). To demonstrate a prolonged inhibition of cell growth, quiescent cells were infected with 6 × 107 virus particles per milliliter; this dose was chosen based on results from the dose-response thymidine incorporation experiment showing approximately 80% to 90% inhibition relative to control (Figure 1A). Five days after treatment, rAd.p21-treated cells showed a significant 3-fold decrease in cell number compared with controls (P<.001, unpaired t test). Treatment with rAd.control did not significantly depress overall cell growth relative to untreated cells(P>.05, unpaired t test), demonstrating a p21-specific effect (Figure 1B).

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Figure 1.

Treatment of rabbit Tenon fibroblasts with rAd.p21 inhibits DNA synthesis and proliferation in vitro. A, Tritiated thymidine incorporation, plotted as a percentage of untreated cells, is inversely correlated with rAd.p21 concentration. No effect was observed with rAd.control treatment. Complete inhibition of incorporation was demonstrated at rAd.p21 concentrations of 1 × 108 P/mL or greater. B, Trypan blue counts of viable cells 5 days after treatment with 6.7 × 107P/mL of rAd.p21 or rAd.control. Treatment with rAd.p21 significantly reduced the proliferation of Tenon fibroblasts in culture (P<.001, unpaired t test), whereas the rAd.control did not(P >.05). Data are given as mean ± SD. rAd.p21 indicates recombinant adenovirus containing the human p21 gene; rAd.control, control recombinant adenovirus; and P/mL, virus particles per milliliter.

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Table Graphic Jump LocationTable 1. Fluorescent-Activated Cell Sorter Analysis of Human Tenon Fibroblasts Treated With rAd.control or rAd.p21*
IN VIVO STUDIES
Characteristics of Transgene Expression

Experiments were conducted to determine the pattern and persistence of transgene expression. Immunohistochemical staining for human p21 protein in rabbit eyes 14 days after rAd.p21 treatment and surgery showed numerous fibroblasts expressing the transgene throughout the flap region (Figure 2B). No p21 staining was evident in eyes treated with BSS (Figure 2A), rAd.control, or mitomycin (data not shown). Other experiments, conducted with the β-galactosidase–expressing vector to identify transduced cells, showed that vascular endothelial and conjunctival epithelial cells were transduced in addition to Tenon fibroblasts. No evidence of p21 expression in scleral fibroblasts, ciliary body epithelial cells, retina, iris, or any cells of the cornea was found using this mode of recombinant adenovirus application(data not shown).

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Figure 2.

Immunohistochemical localization of human p21 protein in cells of treated rabbit eyes 14 days after sclerostomy. A, Nomarski interference photomicrograph of the wound area in the conjunctiva and Tenon capsule of an eye treated with balanced saline solution. No staining is evident. The asterisk indicates a small open area remaining from the surgical bleb that is predominantly filled with fibroblasts. B, Micrograph of the corresponding region in an eye treated with a recombinant adenovirus containing the human p21 gene. Cells lining the open areas of the bleb (asterisks) are positive for human p21 protein. The overlying conjunctival flap (con) is positioned at the top of each image (original magnification ×400).

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The persistence of p21 transgene expression was investigated at the messenger RNA and protein levels. Quantitative reverse transcription–polymerase chain reaction analysis identified transgene-specific transcripts as late as 60 days after treatment, with levels decreasing throughout the experiment(Figure 3A). Enzyme-linked immunosorbent assay analysis of protein fractions from the same eyes showed detectable recombinant protein up to 28 days after infection (day 60 was not evaluated) (Figure 3B), which also decreased with time.

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Figure 3.

In vivo p21 transgene expression persists beyond 30 days. A, Reverse transcription–polymerase chain reaction analysis to detect recombinant adenovirus containing the human p21 gene messenger RNA shows detectable transcripts in homogenates of surgical sites up to at least 60 days after treatment by sponge application. The mean copy number of messenger RNA is calibrated against a standard of known copies of template. B, Enzyme-linked immunosorbent assay analysis indicates that p21 protein persists up to 28 days after treatment (day 60 not done). Protein concentration is indicated as human p21 units, based on an internal standard of purified human p21 protein. Data are given as mean ± SD.

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Slitlamp Examination of Blebs

Rabbits were examined for bleb height at various points after surgery. Figure 4 shows the change in the bleb score during a 30-day period for eyes treated with rAd.p21 compared with PBS containing 3% sucrose and mitomycin. Statistical evaluation of the data during the experiment showed significantly larger blebs for mitomycin- and rAd.p21-treated eyes relative to the eyes treated with PBS containing 3% sucrose (P<.001, unpaired t test), while blebs in the mitomycin-treated rabbits were slightly larger than blebs in the rAd.p21-treated rabbits (P = .03).

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Figure 4.

Bleb scores for rabbit eyes treated with mitomycin, 0.05 mg/mL; rAd.p21; or V-PBS. The mean ± SEM for 6 eyes at each treatment is shown over 30 days from the day of surgery and treatment. Mitomycin-treated eyes exhibited significantly larger blebs relative to rAd.p21(P = .03, unpaired t test) and V-PBS (P<.001) during all stages of the experiment. Eyes treated with rAd.p21 exhibited significantly larger blebs than those treated with V-PBS (P<.001). rAd.p21 indicates recombinant adenovirus containing the human p21 gene; V-PBS, phosphate-buffered saline containing 3% sucrose.

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Histological Evaluation of Wound Healing

The surgical sites were examined 30 days after surgery from sections cut through the surgical site and stained with Gomori trichrome. Figure 5 shows a panel of representative sections from eyes of each treatment group. Eyes treated with either BSS or control virus (recombinant adenoviruses containing either green fluorescent protein or β-galactosidase) exhibited nearly complete scarring over the sclerostomy site, including evidence of new collagen deposition in the scleral gap created by surgery (Figure 5A and B, respectively). In contrast, eyes treated with rAd.p21 had moderate bleb cavities and minimal evidence of new collagen deposition in the sclera(Figure 5C). Eyes treated with mitomycin exhibited large acellular bleb cavities with substantial damage to surrounding tissues, including thinning of the conjunctiva and Tenon layer in the region of the surgery (Figure 5D).

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Figure 5.

Representative Gomori trichrome–stained sections through the surgical sites of rabbit eyes 30 days after surgery. Sections are shown from eyes treated with BSS (A), rAd.GFP (B), rAd.p21 (C), and mitomycin (D). In each micrograph, the cornea is to the right and the site of the sclerostomy is indicated by arrows. The BSS- and rAd.GFP-treated eyes have new collagen deposition (indicated by the light blue–stained material) in the region between the arrows, while this region is open in the eyes treated with rAd.p21 and mitomycin. The eye treated with rAd.p21 also shows the presence of cells in a noncollagenous matrix underlying a fairly robust Tenon capsule and conjunctiva. The bleb appearance of the mitomycin-treated eye shows virtually no evidence of cells and a dramatically thinned conjunctiva, which has allowed the bleb wall to rupture during histological processing. BSS indicates balanced saline solution; rAd.GFP, recombinant adenovirus containing green fluorescent protein; rAd.p21, recombinant adenovirus containing the human p21 gene; and bar, 0.5 mm.

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Sections (range, 5-15) through each surgical site were masked and scored by 2 pathologists for the 2 criteria of fibroproliferation and cellular infiltrate into the surgical site. The consensus score of each criterion, for each eye, is shown in Table 2. For fibroproliferation, most eyes treated with BSS or control virus received scores of 3, indicative of a completely healed wound, while 5 of 6 rAd.p21-treated eyes and 4 of 5 mitomycin-treated eyes received scores of less than 3. On average, the eyes treated with BSS, control adenovirus, or rAd.p21 displayed minimal cellular infiltrate. Eyes treated with mitomycin, however, showed mild to moderate signs of inflammatory cells, including 2 that exhibited an abscess with hypopyon and hyalitis extending from the bleb cavity into the anterior chamber (Figure 6).

Table Graphic Jump LocationTable 2. Fibroproliferation and Cellular Infiltration Scores for Rabbit Eyes Prepared 30 Days After Sclerostomy*
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Figure 6.

Gomori trichrome–stained section of the surgical site of an eye treated with mitomycin. The cornea is to the right of the micrograph and the conjunctiva (con) is at the top. The region of the sclerostomy is indicated with arrows. At the time of humane killing, 30 days after surgery, this eye exhibited severe infiltration of inflammatory cells extending from the Tenon capsule through the sclerostomy and into the anterior and posterior chambers of the eye (original magnification×100).

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Intraocular Pressure

Intraocular pressure measurements taken on awake rabbits with a handheld applanation tonometer exhibited a high degree of variability. In general, the IOP decreased below baseline immediately as a result of the sclerostomy regardless of treatment. Surgical procedures that failed did so within 10 days, as determined by a return to the baseline IOP (data not shown). The data for each treatment are as follows:0

The data were taken directly from the handheld applanation tonometer and not calibrated to true millimeters of mercury for the rabbit eye. The rAd.p21- and the mitomycin-treated eyes showed a trend toward maintaining a reduced IOP during the experiment, although these data do not show a significant decrease over the BSS- or control vector–treated eyes.

Outflow Facility

Figure 7 shows the results of outflow facility measurements in rabbit eyes 14 days after surgery, after the point when BSS-treated eyes were observed to fail based on IOP measurements. The mean ± SD outflow facility of unoperated on rabbit eyes (n = 11) was 0.26 ± 0.04 µL/min per millimeter of mercury. A sclerostomy using BSS or rAd.p21 had no significant (P = .10, Mann-Whitney test) influence on the outflow facility of a normal-tension eye after 14 days, but mitomycin treatment created a significant increase in facility vs unoperated on eyes (P = .02).

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Figure 7.

Outflow facility of normal-tension rabbit eyes 14 days after sclerostomy with mitomycin, rAd.p21, or BSS, compared with unoperated on eyes. The mean ± SEM of a minimum of 4 eyes per group is shown. The BSS- and rAd.p21-treated eyes have no significant increase in outflow facility (P>.20, Mann-Whitney test), while mitomycin causes a significant increase in facility relative to untreated eyes (P = .02). rAd.p21 indicates recombinant adenovirus containing the human p21 gene; BSS, balanced saline solution.

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EFFECT OF rAd.p21 IN PREVENTING WOUND HEALING

Several measurements were taken to judge the effect of p21 gene therapy in controlling wound healing in the rabbit eye. Observations of the bleb characteristics and histological evaluation of the wound indicate that the p21 transgene attenuates fibroproliferation and scarring in the rabbit model at a level comparable to mitomycin-treated eyes. Intraocular pressure measurements were also taken as a measure of performance of the sclerostomies. Although there was a trend toward a decrease in IOP for eyes treated with rAd.p21 and mitomycin, these data were variable and not significantly different from those of eyes treated with BSS or the control virus. Several investigations with the rabbit model have used IOP as an indicator of efficacy for antiproliferative treatments. Jampel and Moon12 showed that paclitaxel (Taxol) and mitomycin were able to prevent wound healing as judged by the change in IOP relative to the unoperated on eye of each animal. Intraocular pressure was also used to evaluate antibodies against transforming growth factor β2 by Cordeiro and coworkers.14 Their study failed to find any effect on IOP in rabbits, although effects on bleb morphologic characteristics were detected. Other studies have questioned the usefulness of IOP as a measure of successful filtration surgery and bleb function in normal-tension rabbit eyes because the ciliary epithelium and residual iris have a tendency to extend forward into the anterior chamber and block the fistula.22 This problem is evident in histological sections taken through the eyes used in this study (Figure 5), which often show the iris either pushing into the fistula or extended toward the cornea.

Our study also included examination of outflow facility in treated compared with unoperated on eyes 14 days after surgery, when control blebs had typically failed (based on IOP measurements). No change in outflow facility was noted for the rAd.p21-treated eyes relative to the BSS-treated eyes or the unoperated on eyes. However, the sclerostomy provided only a small option for outflow in these eyes, which contained an undamaged outflow pathway that was presumably still functioning normally around the entire remaining circumference. It is not surprising that a functioning bleb may not be reflected in these measurements. Mitomycin-treated eyes did show a significant increase in outflow relative to normal eyes. This observation is consistent with studies23 showing an increased risk of hypotonia in human eyes that undergo trabeculectomies using mitomycin. Mitomycin also produced complications in our study that were not observed with rAd.p21. The most noticeable were the increased incidence of inflammatory cells in mitomycin-treated eyes at 30 days after surgery (2 of 5 eyes) and the thinning of the Tenon capsule and conjunctiva in the surgical area (5 of 5 eyes). Although the numbers of cases reported in this study were too few to assign a definitive correlation with mitomycin use, these complications were not observed in any of the other eyes. These data are also consistent with numerous reports2426 that mitomycin treatment can lead to excessively leaky and thin-walled blebs that are prone to late-onset infections.

THE MECHANISM OF ACTION OF THE p21 TRANSGENE

The sequence of events that lead to wound healing includes the proliferation and migration of cells to the site of damage, the deposition of new extracellular matrix, the contraction of the wound margins, and the subsequent programmed death of infiltrating fibroblasts at the end of the scarring process.27 The strategy of using antiproliferative agents to prevent wound healing is to block the proliferation step, which disrupts the subsequent wound-healing cascade. Mitomycin is an alkylating agent that has numerous toxic effects on cells, although its primary mode of action is to cross-link DNA.28,29 Cultured Tenon fibroblasts treated with mitomycin undergo apoptotic cell death,30 and electron microscopic evaluations of mitomycin-treated rabbit conjunctiva and Tenon capsule show extensive tissue damage.14 Previous studies of p21 function show that this protein can also cause apoptotic cell death in some circumstances,31,32 but in most cases it causes cell cycle arrest.68 Treatment of cultured Tenon fibroblasts with rAd.p21 results in reduced DNA synthesis and cell division, while fluorescent-activated cell sorter analysis shows that these cells accumulate in the G1 phase of the cell cycle.

The present in vivo experiments do not conclusively demonstrate that p21 inhibits wound healing by preventing cell division and subsequent extracellular matrix deposition, but Gomori trichrome staining clearly shows reduced collagen deposition and fibroproliferation in the surgical area. To expand these results, experiments are under way to characterize the time course of extracellular matrix deposition and to demonstrate inhibition of cellular proliferation in situ.

In summary, our results demonstrate the p21-specific inhibition of fibroproliferation and wound healing in a rabbit model of glaucoma surgery, which resulted in patent fistulas. These results are comparable to those obtained with mitomycin, but the toxic adverse effects, such as thin filtration blebs and persistent inflammation, observed with mitomycin use are eliminated. Consequently, p21 gene therapy may make an attractive alternative to mitomycin in those undergoing trabeculectomy.

Submitted for publication August 28, 2001; final revision received March 13, 2002; accepted March 20, 2002.

This study was supported by a grant from Canji, Inc, San Diego (Dr Nickells), and by grant EY02698 from the National Eye Institute, Bethesda, Md (Dr Kaufman).

We thank Ken Wills for adenovirus vector creation; Shufen Wen for oversight of polymerase chain reaction/reverse transcription–polymerase chain reaction efforts; the members of the Process Sciences Department, Canji, Inc(Josie Beltran, Doug Cornell, Daniel Giroux, Wendy Hancock, Beth Hutchins, Diane McAllister, Susan Miller, Margarita Nodelman, Thomas Schluep, Anastasia Sofianos, Barry Sugarman, and Suganto Sutjipto) for the production and characterization of all viruses used; Steven Schwartz, MD, for providing human Tenon fibroblasts; Jennifer Seemans, DVM, for her assistance with the rabbit surgical procedures; and Daniel Albert, Morton Smith, T. Michael Nork, MD, and Cassandra Schlamp, PhD, for their constructive comments during the study.

Corresponding author and reprints: Robert W. Nickells, PhD, Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, 1300 University Ave, Room 6640 MSC, Madison, WI 53706-1532 (e-mail: nickells@facstaff.wisc.edu).

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Link to Article
Joshi  USChen  YOKalemkerian  GPAdil  MRKraut  MSarkar  FH Inhibition of tumor growth by p21WAF1 adenoviral gene transfer in lung cancer. Cancer Gene Ther. 1998;5183- 191
Mobley  SRLiu  TJHudson  JMClayman  GL In vitro growth suppression by adenoviral transduction of p21 and p16 in squamous cell carcinoma of the head and neck: a research model for combination gene therapy. Arch Otolaryngol Head Neck Surg. 1998;12488- 92
Link to Article
Nielsen  LLManeval  DC p53 tumor suppressor gene therapy for cancer. Cancer Gene Ther. 1998;552- 63
Schuler  MHermann  RDe Greve  JL  et al.  Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemotherapy for advanced non–small-cell lung cancer: results of a multicenter phase II study. J Clin Oncol. 2001;191750- 1758
Perkins  TWGangnon  RLadd  WKaufman  PLHeatley  GA Trabeculectomy with mitomycin C: intermediate-term results. J Glaucoma. 1998;7230- 236
Jampel  HDMoon  JI The effect of paclitaxel powder on glaucoma filtration surgery in rabbits. J Glaucoma. 1998;7170- 177
Mietz  HChevez-Barrios  PFeldman  RMLieberman  MW Suramin inhibits wound healing following filtering procedures for glaucoma. Br J Ophthalmol. 1998;82816- 820
Link to Article
Cordeiro  MFGay  JAKhaw  PT Human anti–transforming growth factor-β2 antibody: a new glaucoma anti-scarring agent. Invest Ophthalmol Vis Sci. 1999;402225- 2234
Wills  KNManeval  DCMenzel  P  et al.  Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer. Hum Gene Ther. 1994;51079- 1088
Link to Article
Huyghe  BGLiu  XDSutjipto  S  et al.  Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography. Hum Gene Ther. 1995;61403- 1416
Link to Article
Wen  SFXie  LMcDonald  M  et al.  Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models. Cancer Gene Ther. 2000;71469- 1480
Link to Article
Nork  TMPoulsen  GMillecchia  LLJantz  RGNickells  RW p53 regulates apoptosis in human retinoblastoma. Arch Ophthalmol. 1997;115213- 219
Link to Article
Abrams  LSVitale  SJampel  HD Comparison of three tonometers for measuring intraocular pressure in rabbits. Invest Ophthalmol Vis Sci. 1996;37940- 944
Gomori  GL A rapid one-step trichrome. Am J Clin Pathol. 1950;20661
Bárány  EH Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion. Invest Ophthalmol. 1964;2135- 143
Miller  MHGrierson  IUnger  WIHitchings  RA Wound healing in an animal model of glaucoma fistulizing surgery in the rabbit. Ophthalmic Surg. 1989;20350- 357
Suner  IJGreenfield  DSMiller  MPNicolela  MTPalmberg  PF Hypotony maculopathy after filtering surgery with mitomycin C: incidence and treatment. Ophthalmology. 1997;104207- 214
Link to Article
Higginbotham  EJStevens  RKMusch  DC  et al.  Bleb-related endophthalmitis after trabeculectomy with mitomycin C. Ophthalmology. 1996;103650- 656
Link to Article
Greenfield  DSLiebmann  JMJee  JRitch  R Late-onset bleb leaks after glaucoma filtering surgery. Arch Ophthalmol. 1998;116443- 447
Link to Article
Akova  YABulut  SDabil  HDuman  S Late bleb-related endophthalmitis after trabeculectomy with mitomycin C. Ophthalmic Surg Lasers. 1999;30146- 151
Daniels  JTOccleston  NLCrowston  JG  et al.  Understanding and controlling the scarring response: the contribution of histology and microscopy. Microsc Res Tech. 1998;42317- 333
Link to Article
Tomasz  M Mitomycin C: small, fast and deadly (but very selective). Chem Biol. 1995;2575- 579
Link to Article
Paz  MMDas  TATomasz  M Mitomycin C linked to DNA minor groove binding agents: synthesis, reductive activation, DNA binding and cross-linking properties and in vitro antitumor activity. Bioorg Med Chem. 1999;72713- 2726
Link to Article
Crowston  JGAkbar  ANConstable  PHOccleston  NLDaniels  JTKhaw  PT Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts. Invest Ophthalmol Vis Sci. 1998;39449- 454
Kondo  YKondo  SLiu  JBHaqqi  TBarnett  GHBarna  BP Involvement of p53 and waf1/cip1 in γ-irradiation–induced apoptosis of retinoblastoma cells. Exp Cell Res. 1997;23651- 56
Link to Article
Tsao  YPHuang  SJChang  JLHsieh  JTPong  RCChen  SL Adenovirus-mediated p21(WAF1/SDII/CIP1) gene transfer induces apoptosis of human cervical cancer cell lines. J Virol. 1999;734983- 4990

Figures

Place holder to copy figure label and caption
Figure 1.

Treatment of rabbit Tenon fibroblasts with rAd.p21 inhibits DNA synthesis and proliferation in vitro. A, Tritiated thymidine incorporation, plotted as a percentage of untreated cells, is inversely correlated with rAd.p21 concentration. No effect was observed with rAd.control treatment. Complete inhibition of incorporation was demonstrated at rAd.p21 concentrations of 1 × 108 P/mL or greater. B, Trypan blue counts of viable cells 5 days after treatment with 6.7 × 107P/mL of rAd.p21 or rAd.control. Treatment with rAd.p21 significantly reduced the proliferation of Tenon fibroblasts in culture (P<.001, unpaired t test), whereas the rAd.control did not(P >.05). Data are given as mean ± SD. rAd.p21 indicates recombinant adenovirus containing the human p21 gene; rAd.control, control recombinant adenovirus; and P/mL, virus particles per milliliter.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Immunohistochemical localization of human p21 protein in cells of treated rabbit eyes 14 days after sclerostomy. A, Nomarski interference photomicrograph of the wound area in the conjunctiva and Tenon capsule of an eye treated with balanced saline solution. No staining is evident. The asterisk indicates a small open area remaining from the surgical bleb that is predominantly filled with fibroblasts. B, Micrograph of the corresponding region in an eye treated with a recombinant adenovirus containing the human p21 gene. Cells lining the open areas of the bleb (asterisks) are positive for human p21 protein. The overlying conjunctival flap (con) is positioned at the top of each image (original magnification ×400).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.

In vivo p21 transgene expression persists beyond 30 days. A, Reverse transcription–polymerase chain reaction analysis to detect recombinant adenovirus containing the human p21 gene messenger RNA shows detectable transcripts in homogenates of surgical sites up to at least 60 days after treatment by sponge application. The mean copy number of messenger RNA is calibrated against a standard of known copies of template. B, Enzyme-linked immunosorbent assay analysis indicates that p21 protein persists up to 28 days after treatment (day 60 not done). Protein concentration is indicated as human p21 units, based on an internal standard of purified human p21 protein. Data are given as mean ± SD.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.

Bleb scores for rabbit eyes treated with mitomycin, 0.05 mg/mL; rAd.p21; or V-PBS. The mean ± SEM for 6 eyes at each treatment is shown over 30 days from the day of surgery and treatment. Mitomycin-treated eyes exhibited significantly larger blebs relative to rAd.p21(P = .03, unpaired t test) and V-PBS (P<.001) during all stages of the experiment. Eyes treated with rAd.p21 exhibited significantly larger blebs than those treated with V-PBS (P<.001). rAd.p21 indicates recombinant adenovirus containing the human p21 gene; V-PBS, phosphate-buffered saline containing 3% sucrose.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 5.

Representative Gomori trichrome–stained sections through the surgical sites of rabbit eyes 30 days after surgery. Sections are shown from eyes treated with BSS (A), rAd.GFP (B), rAd.p21 (C), and mitomycin (D). In each micrograph, the cornea is to the right and the site of the sclerostomy is indicated by arrows. The BSS- and rAd.GFP-treated eyes have new collagen deposition (indicated by the light blue–stained material) in the region between the arrows, while this region is open in the eyes treated with rAd.p21 and mitomycin. The eye treated with rAd.p21 also shows the presence of cells in a noncollagenous matrix underlying a fairly robust Tenon capsule and conjunctiva. The bleb appearance of the mitomycin-treated eye shows virtually no evidence of cells and a dramatically thinned conjunctiva, which has allowed the bleb wall to rupture during histological processing. BSS indicates balanced saline solution; rAd.GFP, recombinant adenovirus containing green fluorescent protein; rAd.p21, recombinant adenovirus containing the human p21 gene; and bar, 0.5 mm.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 6.

Gomori trichrome–stained section of the surgical site of an eye treated with mitomycin. The cornea is to the right of the micrograph and the conjunctiva (con) is at the top. The region of the sclerostomy is indicated with arrows. At the time of humane killing, 30 days after surgery, this eye exhibited severe infiltration of inflammatory cells extending from the Tenon capsule through the sclerostomy and into the anterior and posterior chambers of the eye (original magnification×100).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 7.

Outflow facility of normal-tension rabbit eyes 14 days after sclerostomy with mitomycin, rAd.p21, or BSS, compared with unoperated on eyes. The mean ± SEM of a minimum of 4 eyes per group is shown. The BSS- and rAd.p21-treated eyes have no significant increase in outflow facility (P>.20, Mann-Whitney test), while mitomycin causes a significant increase in facility relative to untreated eyes (P = .02). rAd.p21 indicates recombinant adenovirus containing the human p21 gene; BSS, balanced saline solution.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Fluorescent-Activated Cell Sorter Analysis of Human Tenon Fibroblasts Treated With rAd.control or rAd.p21*
Table Graphic Jump LocationTable 2. Fibroproliferation and Cellular Infiltration Scores for Rabbit Eyes Prepared 30 Days After Sclerostomy*

References

Levine  AJ p53, the cellular gatekeeper for growth and division. Cell. 1997;88323- 332
Link to Article
El-Deiry  WTokino  TVelculescu  VE  et al.  WAF1, a potential mediator of p53 tumor suppression. Cell. 1993;75817- 825
Link to Article
El-Deiry  WSHarper  JWO'Connor  PM  et al.  WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 1994;541169- 1174
Harper  JWAdami  GRWei  NKeyomarsi  KElledge  SJ The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993;75805- 816
Link to Article
Gartel  ALSerfas  MSTyner  AL p21: negative regulator of the cell cycle. Proc Soc Exp Biol Med. 1996;213138- 149
Link to Article
Erhardt  JAPittman  RN p21WAF1 induces permanent growth arrest and enhances differentiation, but does not alter apoptosis in PC12 cells. Oncogene. 1998;16443- 451
Link to Article
Joshi  USChen  YOKalemkerian  GPAdil  MRKraut  MSarkar  FH Inhibition of tumor growth by p21WAF1 adenoviral gene transfer in lung cancer. Cancer Gene Ther. 1998;5183- 191
Mobley  SRLiu  TJHudson  JMClayman  GL In vitro growth suppression by adenoviral transduction of p21 and p16 in squamous cell carcinoma of the head and neck: a research model for combination gene therapy. Arch Otolaryngol Head Neck Surg. 1998;12488- 92
Link to Article
Nielsen  LLManeval  DC p53 tumor suppressor gene therapy for cancer. Cancer Gene Ther. 1998;552- 63
Schuler  MHermann  RDe Greve  JL  et al.  Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemotherapy for advanced non–small-cell lung cancer: results of a multicenter phase II study. J Clin Oncol. 2001;191750- 1758
Perkins  TWGangnon  RLadd  WKaufman  PLHeatley  GA Trabeculectomy with mitomycin C: intermediate-term results. J Glaucoma. 1998;7230- 236
Jampel  HDMoon  JI The effect of paclitaxel powder on glaucoma filtration surgery in rabbits. J Glaucoma. 1998;7170- 177
Mietz  HChevez-Barrios  PFeldman  RMLieberman  MW Suramin inhibits wound healing following filtering procedures for glaucoma. Br J Ophthalmol. 1998;82816- 820
Link to Article
Cordeiro  MFGay  JAKhaw  PT Human anti–transforming growth factor-β2 antibody: a new glaucoma anti-scarring agent. Invest Ophthalmol Vis Sci. 1999;402225- 2234
Wills  KNManeval  DCMenzel  P  et al.  Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer. Hum Gene Ther. 1994;51079- 1088
Link to Article
Huyghe  BGLiu  XDSutjipto  S  et al.  Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography. Hum Gene Ther. 1995;61403- 1416
Link to Article
Wen  SFXie  LMcDonald  M  et al.  Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models. Cancer Gene Ther. 2000;71469- 1480
Link to Article
Nork  TMPoulsen  GMillecchia  LLJantz  RGNickells  RW p53 regulates apoptosis in human retinoblastoma. Arch Ophthalmol. 1997;115213- 219
Link to Article
Abrams  LSVitale  SJampel  HD Comparison of three tonometers for measuring intraocular pressure in rabbits. Invest Ophthalmol Vis Sci. 1996;37940- 944
Gomori  GL A rapid one-step trichrome. Am J Clin Pathol. 1950;20661
Bárány  EH Simultaneous measurement of changing intraocular pressure and outflow facility in the vervet monkey by constant pressure infusion. Invest Ophthalmol. 1964;2135- 143
Miller  MHGrierson  IUnger  WIHitchings  RA Wound healing in an animal model of glaucoma fistulizing surgery in the rabbit. Ophthalmic Surg. 1989;20350- 357
Suner  IJGreenfield  DSMiller  MPNicolela  MTPalmberg  PF Hypotony maculopathy after filtering surgery with mitomycin C: incidence and treatment. Ophthalmology. 1997;104207- 214
Link to Article
Higginbotham  EJStevens  RKMusch  DC  et al.  Bleb-related endophthalmitis after trabeculectomy with mitomycin C. Ophthalmology. 1996;103650- 656
Link to Article
Greenfield  DSLiebmann  JMJee  JRitch  R Late-onset bleb leaks after glaucoma filtering surgery. Arch Ophthalmol. 1998;116443- 447
Link to Article
Akova  YABulut  SDabil  HDuman  S Late bleb-related endophthalmitis after trabeculectomy with mitomycin C. Ophthalmic Surg Lasers. 1999;30146- 151
Daniels  JTOccleston  NLCrowston  JG  et al.  Understanding and controlling the scarring response: the contribution of histology and microscopy. Microsc Res Tech. 1998;42317- 333
Link to Article
Tomasz  M Mitomycin C: small, fast and deadly (but very selective). Chem Biol. 1995;2575- 579
Link to Article
Paz  MMDas  TATomasz  M Mitomycin C linked to DNA minor groove binding agents: synthesis, reductive activation, DNA binding and cross-linking properties and in vitro antitumor activity. Bioorg Med Chem. 1999;72713- 2726
Link to Article
Crowston  JGAkbar  ANConstable  PHOccleston  NLDaniels  JTKhaw  PT Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts. Invest Ophthalmol Vis Sci. 1998;39449- 454
Kondo  YKondo  SLiu  JBHaqqi  TBarnett  GHBarna  BP Involvement of p53 and waf1/cip1 in γ-irradiation–induced apoptosis of retinoblastoma cells. Exp Cell Res. 1997;23651- 56
Link to Article
Tsao  YPHuang  SJChang  JLHsieh  JTPong  RCChen  SL Adenovirus-mediated p21(WAF1/SDII/CIP1) gene transfer induces apoptosis of human cervical cancer cell lines. J Virol. 1999;734983- 4990

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