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Adenovirus-Mediated Gene Therapy Using Human p21WAF-1/Cip-1 to Prevent Wound Healing in a Rabbit Model of Glaucoma Filtration Surgery
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
Arch Ophthalmol. 2002;120:941-949.
ABSTRACT
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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  -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.
INTRODUCTION
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).2-4 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 studies6-8
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.12-14
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.
MATERIALS AND METHODS
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 -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 x 107-3 x 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 x 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 x 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 x 4 x
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.
RESULTS
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 x 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 x 108 P/mL or greater. B, Trypan blue
counts of viable cells 5 days after treatment with 6.7 x 107
P/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 1. Fluorescent-Activated Cell Sorter Analysis of Human Tenon
Fibroblasts Treated With rAd.control or rAd.p21*
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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 -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 x400).
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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 -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).
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Table 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
x100).
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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:
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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COMMENT
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 2 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 reports24-26
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.6-8
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.
AUTHOR INFORMATION
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{at}facstaff.wisc.edu).
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.
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