Abstract
purpose. To compare the effects of the three human isoforms of transforming
growth factor (TGF)-β in vivo using a mouse model of conjunctival
scarring, both in normal eyes and after treatment with MMC, with a view
to delineating the role of this growth factor in glaucoma filtration
surgery.
methods. Application of recombinant human TGF-β was assessed in a prospective,
randomized study of mouse conjunctival scarring, in which
subconjunctival TGF-β1, -β2, and -β3 (all 10−9 M)
were compared with control (phosphate-buffered saline [PBS] carrier)
and mitomycin C (MMC; 0.4 mg/ml) treatment at 6 hours, and 1, 3, and 7
days after surgery (six eyes/treatment/time point). Effects of TGF-β2
on eyes previously treated with MMC were also assessed. Histologic
studies of enucleated eyes were performed to analyze development of the
scarring response, extracellular matrix deposition, and the
inflammatory cell profile.
results. All three isoforms of TGF-β behaved in a similar manner in vivo,
being associated with a rapid-onset and exaggerated scarring response
compared with control and MMC treatment. TGF-β–treated eyes showed
evidence of an earlier peak in inflammatory cell activity
(P < 0.05) and increased collagen type III
deposition (P < 0.05). TGF-β2 treatment
significantly stimulated scarring after MMC application
(P < 0.05).
conclusions. TGF-β1, -β2, and -β3 appear to have similar actions in vivo and
stimulate the conjunctival scarring response. Application of TGF-β2
modified the effects of MMC. All TGF-β isoforms may be potent
modulators of the conjunctival scarring response. These studies
indicate that TGF-β2 may naturally modify the antiscarring effects of
antimetabolites such as MMC in glaucoma filtration
surgery.
The conjunctival scarring response is a major determinant of
morbidity and visual prognosis in a wide spectrum of ocular diseases
including cicatricial conditions (e.g., trachoma and pemphigoid) and
those in which the results of treatment depend on the healing response
after surgery, such as glaucoma. The commonest cause of failure of
glaucoma filtration surgery is the occurrence of subconjunctival
scarring at the bleb and sclerostomy sites,
1 2 with
increased scar deposition being associated with poor control of
postoperative intraocular pressure.
Although the role of growth factors in conjunctival scarring is
believed to be important, that of transforming growth factor-β
(TGF-β), the most potent growth factor involved in wound healing
throughout the body,
3 4 5 6 is unclear. There are three
isoforms of TGF-β found in humans. Of these, TGF-β1 and -β2 are
known to stimulate greatly the dermal scarring
response.
4 5 6 7 8 9 10 The actions of the third isoform, TGF-β3,
in wound healing are less well established, with some studies
suggesting that it may actually inhibit in vivo
scarring.
4 11
Although the effects of exogenous TGF-β have been studied in skin,
the actions of all three isoforms after administration in the eye have
not been examined to date. However, it is known that compared with
TGF-β1 and -β3, TGF-β2 is the predominantly expressed ocular
isoform, having been identified in normal and diseased
eyes
12 13 and implicated in the pathogenesis of several
ocular scarring diseases such as proliferative vitreoretinopathy and
cataract formation.
14 15 More recent work, however, has
suggested TGF-β1 and -β3 may be important in the fibrosis occurring
in the cicatrizing disease, ocular pemphigoid.
16
The conjunctival scarring response in glaucoma filtration surgery is
thought to be greatly influenced by the passage of aqueous through the
surgical site, and in particular, growth factors in the aqueous such as
TGF-β.
17 However, compared with the other growth factors
found in aqueous humor, TGF-β2 has been shown to be the most
potent
18 and is significantly raised in glaucomatous
eyes.
19 In the light of these findings, our study
specifically investigates the effects of TGF-β on the conjunctival
scarring response.
Using our recently established model of conjunctival scarring in the
mouse eye,
20 we have compared the effects of all three
TGF-β isoforms. The model is unique because unlike fistulizing
glaucoma surgery, which causes a complex, dynamically changing wound
environment involving the sclera, the conjunctiva, the aqueous humor,
its various cytokines and chemical mediators, it consists of a
wound-healing response that is specifically localized to the
subconjunctival space. We have investigated the conjunctival scarring
response induced by TGF-β in the mouse eye, using histologic
techniques that identify collagen, elastic-related fibers, and
inflammatory cells and have compared this to control and the commonly
used modulating agent mitomycin C (MMC). In addition, we have assessed
the effects of TGF-β2 on MMC-treated eyes.
The exogenous TGF-βs used in these studies were all recombinant
human proteins. TGF-β2 and -β3 were generous gifts of Ciba Geigy,
Switzerland, and TGF-β1 was a gift from Professor Gregory Schultz,
University of Florida, Gainesville. All TGF-β injections were
reconstituted from stock solutions of the active form (1 mg/ml) in
phosphate buffered saline (PBS) containing 1% bovine serum albumin
(Sigma) at a concentration of 10−9 M. PBS was
used as the carrier and the control. Mitomycin-C (MMC, Kyowa, Essex,
United Kingdom) was used at the clinically used concentration of 0.4
mg/ml.
All enucleated eyes were prepared for histologic analysis after
fixation for 24 hours with 10% buffered formaldehyde and embedded
whole in paraffin wax. Development of scar tissue was studied in
sequential 5-μm thick sections using the following special stains:
hematoxylin and eosin to assess cellularity, van Gieson and picrocirius
red (viewed under polarized light) to demonstrate collagen deposition,
and aldehyde fuchsin for elastic and elaunin fibers together with
oxidation-aldehyde fuchsin for additionally demonstrating oxytalan
fibers. In addition, immunofluorescence was used to demonstrate
collagen types I and III. Primary antibodies were goat anti-mouse
collagen I (1:25 dilution, Europath, Southern Biotechnology) and chick
anti-mouse collagen III antibodies (1:25 dilution, Europath, Southern
Biotechnology) with a secondary fluorescein isothiocyanate
(FITC)-labeled chick anti-goat and rabbit anti-chick IgG antibodies
(1:100 dilution, Serotec). Photographs were taken at an
excitation–emission wavelength of 490 and 525nm.
Lymphocyte and macrophage identification was performed using the
lymphocyte CD3 rat anti-human antibody (1:50 dilution, Serotec) and
macrophage F4/80 rat anti-mouse antibody (1:20 dilution, Serotec) as
primary antibodies incubated for 2 hours, followed by a further 2-hour
incubation with a FITC-labeled goat anti-rat IgG secondary antibody
(1:100 dilution, Serotec). Fibroblasts were identified using
phalloidin, which stains the fibroblast cytoskeleton.
21 Briefly, sections were soaked in Tris-buffered saline (TBS; 25 mM Tris,
140 mM NaC1, 3 mM KCl [pH 7.4]) before a 2-hour incubation in
FITC-phalloidin (Sigma) at 2.5 μg/ml in TBS. Sections were then
rinsed and mounted in PBS and viewed by fluorescence microscopy.
As expected, there was a significant reduction in total
cellularity associated with MMC treatment compared with control and all
TGF-β-treated eyes (days 3 and 7; P < 0.05). In
comparison with control, MMC treatment significantly inhibited
macrophage and lymphocyte levels on day 1 and fibroblast activity on
days 1, 3, and 7 (P < 0.05). Macrophage activity was
significantly reduced by MMC treatment compared with TGF-β1, -β2,
and -β3 at 6 hours (P < 0.05), and there was a
significant difference in lymphocyte and fibroblast levels in MMC and
TGF-β2-treated eyes at 6 hours and 7 days, respectively
(P < 0.05).
A reduction in extracellular matrix components was also associated with
MMC treatment. Compared with control, this was significant on days 3
and 7 for collagen III (P < 0.05), day 7 for collagen
I (P < 0.05), day 3 for elastic fiber, and day 7 for
picrocirius red staining (P < 0.05). Differences among
MMC and TGF-β1, -β2, and -β3 treatments were present with a
reduction in collagen III on days 3 and 7 (P < 0.05),
elastic fibers at 6 hours and on day 3 (P < 0.05), and
picrocirius red staining on day 7.
Our studies have shown that all three human TGF-β isoforms, when
applied exogenously and at the same concentration, produced a similar
conjunctival scarring response. This response was characterized by an
earlier and more pronounced peak in inflammatory cell activity with
evidence of enhanced fibroblast activity in TGF-β treatment groups
compared with control. In addition, TGF-β2 was associated with
increased collagen III deposition, although no significant difference
between TGF-β and control groups was demonstrated in extracellular
matrix architecture.
Little is known about the effect of exogenous TGF-β in conjunctival
scarring. However, elsewhere in the eye, TGF-β has been advocated as
a biologic chorioretinal “glue” for use in repairing retinal
tears
23 and macular holes.
24 Glaser et al.
24 suggested that visual acuity after TGF-β2 treatment
in macular hole surgery significantly improved in a dose-related manner
(range, 0.28–5.32 × 10
−7 M TGF-β2).
Early work by Roberts et al.
10 showed that a
subcutaneous injection of TGF-β1 and -β2 (0–16 ×
10
−7 M) in newborn mice, stimulated the formation of
granulation tissue, associated with induction of angiogenesis,
increased fibroblast number, and collagen deposition. TGF-β
administration into the peritoneum has also been shown to induce
fibrosis.
25 Williams et al.
25 demonstrated
that application of 16 × 10
−8 M TGF-β1 after
surgical injury to the uterine horns in rats, significantly increased
the number of adhesions formed after surgery, with evidence of an
increased number of inflammatory cells and fibroblasts,
histologically.
25
Application of TGF-β2 (4 and 40 × 10
−8 M) on
healing fractures in the rabbit, demonstrated that at the higher dose,
TGF-β2 promoted callus formation in stable conditions.
26 However, in unstable conditions, TGF-β2 was found to be opposite in
that they retarded and reduced bone and cartilage formation in the
callus. TGF-β2 was not found to accelerate fracture healing. This is
suggestive that the actions of TGF-β2 are determined by its
extracellular environment.
The effects of exogenous TGF-β application have also been studied in
several models of dermal wound healing. Shah et al.
22 have
demonstrated that exogenous application of TGF-β1 to a linear
incisional wound in rat skin affects the response in a dose-dependent
manner (range, 0.8–20 × 10
−9 M). At 4 and 20 × 10
−9 M concentrations of TGF-β1, wounds showed
increased vascularity and extracellular matrix deposition compared with
controls, with evidence of scarring. However, no significant difference
in cellularity was noted between TGF-β1 and control groups.
In one of the few studies comparing the in vivo effects of all three
TGF-β isoforms, Shah et al.
4 applied exogenous TGF-β1,
-β2, and -β3 to the same incisional rat model of dermal scarring
described above. All isoforms were assessed at different concentrations
of 0.04, 0.4, 4, and 20 × 10
−9 M. Differences
between the isoforms were noted at 4 and 20 × 10
−9 M
in increased fibronectin in association with TGF-β1 and -β2 only,
increased collagen fibril organization with TGF-β3 only, decreased
monocyte and macrophage profile of wounds treated with TGF-β3
(compared with TGF-β1 and -β2 which were similar to control), and
increased collagen I and III in TGF-β1 and -β2 treatments only. All
isoforms were associated with increased vascularity and angiogenesis.
The authors suggest that TGF-β3 inhibited scarring and promoted
better collagen organization, compared with TGF-β1 and -β2, which
stimulated dermal scarring.
However, Cox
11 has shown that TGF-β3 application, both
in thermal wounds in mice and incisional and second-intention wounds in
rats, stimulates the cutaneous scarring response. This stimulation by
TGF-β3, similar to that associated with TGF-β1 and -β2, resulted
in accelerated re-epithelialization, increased fibroblast and phagocyte
activity, and increased protein, DNA, and collagen production.
Our results, similar to those of Cox,
11 suggest that in
conjunctival scarring all three TGF-β isoforms have similar
stimulatory effects. This is supported by our data from in vitro work
in our laboratory investigating effects of TGF-β on conjunctival
fibroblasts.
27 The TGF-β used in this study was 100%
active. This is different from cellular TGF-β, which, when produced,
is secreted in a latent form that has to be activated into a mature,
active form. It is known that only between 22% and 61% of TGF-β
found in aqueous is in its active rather than its latent form
17 28 and the average concentration of active TGF-β2 in normal
aqueous is between 0.73 and 10.98 × 10
−11 M compared
with 10
−9 M, a much higher concentration, used in our
study.
Using the mouse model of conjunctival scarring, we have shown that
TGF-β2 applied to mice conjunctiva after MMC treatment modulated and
significantly reversed the antiscarring effects of MMC. Specifically,
this was demonstrated by TGF-β2 treatment stimulating cellular
activity including macrophages, lymphocytes, and fibroblasts compared
with either control or MMC treatment alone. In addition, TGF-β2
treatment was associated with increased collagen type I and III
deposition.
The use of TGF-β2 as a modulating agent to counteract effects of MMC
treatment, has been shown by other investigators.
29 30 Doyle et al.
30 demonstrated that a single peribleb
injection of 4 × 10
−7 M TGF-β2 effectively treated
50% of bleb leaks deliberately induced in a rabbit model of
MMC-assisted filtration surgery. Histologic examination revealed
increased peribleb cellularity and denser collagen deposition in
TGF-β eyes compared with control eyes.
The present mouse study also shows the possible use of TGF-β2 as a
modulator of the potent antiscarring effects of conjunctival MMC. This
is important because although MMC has been shown to be highly effective
as an adjuvant treatment for preventing postoperative scarring in
glaucoma filtration surgery, its use is associated with several
complications. These include the production of thin, avascular cystic
blebs with the attendant risks of persistent hypotony, bleb leaks, and
endophthalmitis. TGF-β2 may offer another therapeutic strategy for
enhancing healing after MMC use, although the method of its
instillation would be very important. Our studies on MMC bleb
sizes
31 suggest that a peribleb injection (as administered
by Doyle et al.
30 ) would not be suitable, because leaking
blebs are often thin-walled cystic blebs, with an increase in
cellularity around the bleb itself. A peribleb injection of TGF-β2
would further increase the degree of peribleb cellular activity
contributing to an increase in surrounding scar tissue fibrosis and
contraction, which in turn would produce even smaller blebs with
thinner walls. It would seem more appropriate that TGF-β2 be applied
either transconjunctivally or with an intrableb injection. Obviously,
these methods need further investigation.
In summary, our results show that all three TGF-β human
isoforms have similar actions in subconjunctival scarring in vivo. They
each stimulate the scarring response in an exaggerated and rapid manner
compared with control and MMC. Application of TGF-β2 modulated the
antiscarring effects of MMC. This may be an important clinical finding
and suggests that TGF-β is a potent modulator of the conjunctival
scarring response and may naturally modify the antiscarring effects of
antimetabolites such as MMC in glaucoma filtration surgery.
Reprint requests: M. Francesca Cordeiro, Wound Healing Research and Glaucoma Units, Department of Pathology, Moorfields Eye Hospital and Institute of Ophthalmology, Bath Street, London EC1V 9EL UK.
Supported in part by Wellcome Trust Vision Research Fellowship, United
Kingdom; and Deutsche Forschungsgemeinschaft and Deutsche Retinitis
Pigmentosa Vereinigung.
Submitted for publication December 3, 1998; revised March 24, 1999;
accepted April 8, 1999.
Proprietary interest category: N.
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