Download Role of transforming growth factor β in conjunctival scarring*

Clinical Science (2003) 104, 181–187 (Printed in Great Britain)
GLAXOSMITHKLINE/MRS PAPER
Role of transforming growth factor β in
conjunctival scarring*
M. Francesca CORDEIRO
Department of Pathology, Moorfields Eye Hospital and Institute of Ophthalmology, Bath Street, London EC1V 9EL, U.K.,
and Department of Glaucoma, Moorfields Eye Hospital and Institute of Ophthalmology, Bath Street, London EC1V 9EL, U.K.
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Glaucoma is the major cause of irreversible blindness throughout the world. Of all of the
treatments that are available at present, the most effective appears to be surgery ; however,
excessive conjunctival scarring can lead to surgical failure. In the last decade, the introduction of
the anti-metabolites mitomycin-C and 5-fluorouracil as anti-scarring treatments have greatly
improved the results of glaucoma surgery, but these agents are associated with complications
that can potentially result in blindness. A possible target for a more physiological approach to
anti-scarring is transforming growth factor β. This review examines the role of transforming
growth factor β in conjunctival scarring and discusses promising new ways of modifying its
activity.
INTRODUCTION
Glaucoma is characterized by the development of a
specific pattern of optic neuropathy and visual field loss.
It is the most important cause of irreversible blindness in
both the developed and developing world, accounting for
approx. 15 % of all blindness and over 500 000 new cases
each year [1–3]. The treatment of this disease is directed
towards the reduction of intraocular pressure (the main
identifiable risk factor in glaucoma) [4] and includes
topical medication and laser and surgical modalities. Of
all available therapies, surgery has been shown to be the
most effective [5,6] ; however, glaucoma filtration surgery
does not always work – the most common cause of
failure being the occurrence of conjunctival scarring at
the surgical site [7,8].
Transforming growth factor β (TGF-β) is known to be
the most potent growth factor involved in wound healing
throughout the body [9–12] ; however its role in the
conjunctival wound-healing response has, to date, not
been elucidated. There are three isoforms of TGF-β in
humans. Of these, TGF-β1 and TGF-β2 are known to
greatly stimulate the dermal scarring response [10–16].
The actions of the third isoform, TGF-β3, in wound
healing are less well established, with some studies
suggesting that it may actually inhibit scarring in vivo
[10,17].
Although the effects of exogenous TGF-β have been
studied in skin, the actions of all three isoforms following
administration in the eye, have not been examined
previously ; however, it is known that TGF-β2 is the
major ocular isoform, having been identified in normal
and diseased eyes [18,19] and implicated in the pathogenesis of several ocular scarring diseases such as
proliferative vitreoretinopathy and cataract formation
[20,21]. More recent work has suggested that TGF-β1
and TGF-β3 are important in the cicatrizing disease,
ocular pemphigoid [22].
In the light of these findings, and using both in vivo
and in vitro models, we investigated the expression of
TGF-β during normal conjunctival healing, compared
the effects of all three TGF-β isoforms in vivo and in
* This paper was presented in part at the GlaxoSmithKline\MRS Young Investigator session at the MRS Meeting, Royal College
of Physicians, London, 19 May 1999.
Key words : anti-scarring therapy, conjunctival scarring, glaucoma, transforming growth factor β.
Abbreviations : HTF, human Tenon’s fibroblast ; TGF-β, transforming growth factor β ; MMC, mitomycin-C.
Correspondence : Dr Francesca Cordeiro (e-mail m.cordeiro!ucl.ac.uk).
# 2003 The Biochemical Society and the Medical Research Society
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M. F. Cordeiro
vitro, and studied whether TGF-β could be targeted for
modulation during the conjunctival wound-healing response.
EXPRESSION OF TGF-β IN CONJUNCTIVAL
SCARRING IN VIVO
Using a mouse model of conjunctival scarring, we have
previously developed and characterized [23,24], we
have investigated the expression of TGF-β1, TGF-β2 and
TGF-β3, and found that all three isoforms are expressed
during the conjunctival wound-healing response. TGFβ2 protein was the predominant isoform demonstrated in
both control and treated eyes, being present in the
basement membrane of conjunctival epithelium and
being distributed to varying degrees in the conjunctival
stroma, sclera and corneal stroma. Control eyes showed
increased TGF-β2 activity in the limbal area. In the conjunctival scarring model, TGF-β2 activity in the surgically wounded area was apparent on day 2 and increased
to a peak on day 7, being distributed in a linear pattern
in the stroma (Figure 1).
Studies of TGF-β gene expression using in situ hybridization demonstrated a temporal pattern. TGF-β2
mRNA was found to be the predominant form, with
expression peaking at day 1, and appeared localized to
granulocytes. TGF-β1 and TGF-β3 had maximal expression at day 2 in lymphocytes and mononuclear cells,
although there was also evidence of it on day 1 in
platelets. TGF-β3 mRNA was present, but in much
smaller amounts than the other two isoforms (Table 1).
(a)
(b)
(c)
(d)
(e)
(f )
Figure 1
TGF-β1, TGF-β 2 and TGF-β3 are expressed during the conjunctival scarring response
TGF-β protein expression is demonstrated by immunostaining with diaminobenzidine (dark brown), at days 2 (a–c) and 7 (d–f). TGF-β1 (a,d) TGF-β2 (b,e) and
TGF-β3 (c,f). TGF-β2 activity is predominant with maximal expression found at day 7. Sections are also stained with haematoxylin (purple). Magnification, i25.
Table 1
Immunodistributions of TGF-β isoforms during the conjunctival scarring response
k, staining not detectable ; j, faint staining ; jj, moderate ; jjj, strong immunoreactivity.
Conjunctival bleb area
Control
Day 1
Day 2
Day 3
Day 7
Day 14
Protein
TGF-β1
TGF-β2
TGF-β3
j/k
j
j/k
j
jj
j
j
jj
j
j
jj
j
jj
jjj
jj
j
jjj
j
mRNA
TGF-β1
TGF-β2
TGF-β3
k
j/k
k
j
jjj
j
jj
jj
jj
j
j
j
j/k
j
k
j/k
j
j
Platelets
Granulocytes
Platelets
Macrophages
Lymphocytes
Macrophages
Lymphocytes
Fibroblasts
Fibroblasts
Cellular profile
TGF-β1
TGF-β2
TGF-β3
# 2003 The Biochemical Society and the Medical Research Society
TGF-β in conjunctival scarring
EFFECT OF EXOGENOUS TGF-β ON
CONJUNCTIVAL SCARRING IN VIVO AND
IN VITRO
Using several different in vitro assays, we found that all
three isoforms of TGF-β behaved in a similar manner in
vitro [25]. They each stimulated human Tenon’s fibroblast (HTF)-mediated collagen contraction, proliferation
and migration, with a characteristic concentrationdependent response, with peak activities at 10−* M (TGFβ1), 10−"# M (TGF-β2) and 10−* M (TGF-β3), which
were significantly different from controls (P 0.05). At
concentrations above and below peak activities, HTF
activity was reduced, which demonstrated biphasic effects of TGF-β. HTF proliferation was not detected in
three-dimensional lattices, with either bromodeoxyuridine uptake or Ki67 immunohistochemistry techniques.
Chequerboard analysis of TGF-β2 activity suggested that
migration of HTF was mainly a result of chemotaxis.
Likewise, we found that all three isoforms of TGF-β
behaved in a similar manner in vivo, using the same
mouse model of conjunctival scarring as above [23].
Exogenous TGF-β application was associated with a
rapid-onset and exaggerated scarring response compared
with controls and with treatment with the current antiscarring antiproliferative agent, mitomycin-C (MMC).
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). We found that
TGF-β2 treatment significantly stimulated scarring after
MMC application (P 0.05).
TARGETING TGF-β IN CONJUNCTIVAL
SCARRING IN VITRO
Using a new antibody that was prepared using recombinant techniques by Cambridge Antibody Technology
(CAT, Melbourn, Cambridgeshire, U.K.) [26], we used
several in vitro assays to assess its ability to neutralize
TGF-β2\HTF-mediated activity[27]. This antibody was
designed to specifically target human TGF-β2. We found
the antibody effectively inhibited TGF-β2-mediated conjunctival scarring activity in vitro at similar IC values
&!
[HTF proliferation, 0.13 µg\ml (0.885 nM) ; HTF migration, 0.092 µg\ml (0.627 nM) ; HTF-mediated contraction, 0.12 µg\ml (0.818 nM)].
MODULATING TGF-β ACTIVITY IN VIVO
The ability of the anti-TGF-β2 antibody described in the
previous section to inhibit conjunctival scarring in an in
vivo model [28] of glaucoma surgery was assesed. We
found that application of the anti-TGF-β2 antibody
significantly improved glaucoma surgery outcome in an
animal model of aggressive conjunctival scarring compared with controls [27] ; it also significantly prolonged
surgical survival compared with the control (log rank
statistics, P l 0.0291). Surgical survival was measured by
the persistence of a raised area of conjunctiva, called a
‘ bleb ’, which is caused by aqueous fluid draining from
the anterior chamber to the subconjunctival space in the
eye via a surgically created channel. Any scarring at
the surgical wound site impedes the flow of aqueous
material, which prevents bleb formation. Treatment with
the antibody was associated with an elevated, diffuse,
fleshy-looking bleb, compared with the flat, scarred bleb
in the control group. Histologically, the TGF-β2 antibody appeared to be associated with significantly reduced
scarring activity at a microscopic level, compared with
controls.
Based on our results, a Phase 1 clinical trial of this antiTGF-β2 antibody in primary glaucoma filtration surgery
in patients at Moorfields Eye Hospital, London, was
performed ; no serious adverse effects were noted and
there was evidence of good tolerance and safety [29]. A
Phase IIb multicentre trial is in progress at present
and a Phase III trial has commenced recently.
Finally, another possible strategy that we have investigated is using antisense oligonuclotides to inhibit TGF-β
gene expression. Historically, the delivery of antisense
oligonuclotides into target cells or the cell nucleus has
been problematical, despite the advances in viral and nonviral gene delivery systems. We have shown that a novel
antisense oligonucleotide to TGF-β1 (ISIS Pharmaceuticals, Carlsbad, CA, U.S.A.), which contains both a
phosphorothioate backbone and a 2h-methoxyethyl sugar
modification to increase nuclease stability and antisense
potency [30,31], can reduce conjunctival scarring, using
our mouse model of conjunctival scarring, and that an
antisense oligonucleotide to TGF-β2 (ISIS Pharmaceuticals) can effectively improve surgical outcome in our
rabbit model of glaucoma surgery after only a single
administration at the time of surgery [32].
DISCUSSION
Our results show for the first time that all three isoforms
of TGF-β are expressed in the conjunctiva, although
TGF-β2 appears to be the predominant isoform both in
normal eyes and during the conjunctival wound-healing
response. We have demonstrated that TGF-β1, TGF-β2
and TGF-β3 have similar actions when stimulating the
conjunctival scarring response in vivo and in vitro. We
have also shown that inhibiting TGF-β2 activity can
effectively reduce conjunctival scarring and improve the
outcome of glaucoma filtration surgery.
In our study, we have shown that the TGF-β2 protein
is predominantly expressed in the conjunctiva, being
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M. F. Cordeiro
found normally in the basement membrane of conjunctival epithelium, the conjunctival stroma, the scleral
and corneal stroma, and with increased activity in the
limbal area. During the conjunctival scarring response,
we demonstrated a spatial and temporal relationship
between all three TGF-β isoforms, with peak TGF-β2
mRNA expression at day 1, compared with TGF-β1 and
TGF-β3 mRNA expression, which though less than that
of TGF-β2, peaked on day 2. Staining for TGF-β2 in the
wound area was much stronger than that for either of
the two other isoforms, and reached maximal activity
on day 7. Interestingly, the immunoreactive product was
distributed in a linear, ‘ extracellular network ’ pattern
in the conjunctival stroma, which suggests the presence
of sheets of connective tissue [19,33]. Both TGF-β1
and TGF-β3 were found in the wound area, but in less
significant amounts than TGF-β2.
It has been reported previously that TGF-β2 is the
most prominently expressed isoform in the eye [18],
having being detected in the conjunctival stroma, superficial limbal epithelial cells, ciliary processes, ciliary body
muscles and scleral stroma adjacent to the pars plana [19].
In comparison, only weak staining for TGF-β1 has been
demonstrated in the superficial limbal epithelial cells and
in ciliary processes, with TGF-β3 present only in white
blood cells.
Studies of the expression of the different TGF-β
isoforms in skin suggest spatial differences in isoform
activity during wound healing [11,17,34]. Levine et al.
[11] showed early expression of TGF-β2 and TGF-β3 in
the inflammatory phase, but predominantly TGF-β1
in the proliferating and remodelling phases. Frank et al.
[34] who demonstrated maximal TGF-β1 expression in
the early phases, a relatively constant amount of TGF-β2
activity, and that TGF-β3 expression was greatest in the
proliferating phase of the wound-healing response. In
addition to being involved in skin-healing pathology, all
three isoforms have been shown to have different
activities during embryogenesis [35,36].
The reasons why three homologous TGF-β isoforms
exist in humans is still not fully understood. One possible
explanation might be that the differential patterns of
expression rely on co-ordination of each of the three
isoforms as mediators of mesenchymal–epithelial interactions ; hence in the skin, TGF-β2 and TGF-β3 affect
epithelialization and keratocyte migration, while the
effects of TGF-β1 are predominantly dermal and involve
fibroblast functions [17]. The conjunctival scarring model
used in this project does not involve a breach in the
epithelium. Hence the conjunctival stroma is the target
tissue, which appears, from this work and that of Pasquale
et al. [19], to primarily involve the TGF-β2 isoform. The
mouse model in these studies is different from those
involving excisional wounds and epithelium disruption
[11,34] and therefore does not really investigate TGF-β
isoform mesenchymal–epithelial interactions.
# 2003 The Biochemical Society and the Medical Research Society
It appears from our findings and that of others that
the TGF-β profile in scarring very much depends on
the normal pattern of expression of TGF-β isoforms
in tissue. Hence, since TGF-β2 is predominantly expressed in the eye, and more specifically in the conjunctival stroma, activity of this isoform should be
expected to be greater than the other isoforms during
the wound-healing response. Production of all three
isoforms does occur in the early stages of scarring because the cell population at the site of the wound changes,
i.e. inflammatory cells locally producing the different
TGF-β isoforms. Hence, mRNA expression in our model
was maximal during the inflammatory phase of the scarring response. After the proliferative phase, however,
an attempt is made to restore local architecture via the
processes of remodelling. This is reflected in increased
expression of the TGF-β protein, and in particular,
that of TGF-β2, which is probably expressed by local
fibroblasts secreting newly deposited extracellular
matrix.
All three isoforms of TGF-β stimulate in vitro fibroblast activity, as demonstrated by their effects on assays
of fibroblast mediated-collagen contraction, fibroblast
proliferation and fibroblast migration, and suggesting
that TGF-β has a stimulatory effect on conjunctival
scarring. This stimulation occurs in a biphasic, concentration-dependent manner, with different peak activities
associated with different fibroblast functions. This is
further confirmed by our findings from in vivo experiments, which showed that all three isoforms, when
applied exogenously and at the same concentration,
produced a similar conjunctival scarring response,
characterized by an earlier and more pronounced peak in
inflammatory cell activity with evidence of enhanced
fibroblast activity and increased collagen III deposition
in TGF-β treatment groups compared with controls.
The implications of different peak activities of TGF-β
induced fibroblast functions may be explained physiologically. In a wound environment, the two early
functions of fibroblasts are migration and proliferation.
TGF-β is initially released by inflammatory cells and
platelets at the wound site. At relatively low concentrations, it can act as a stimulant for fibroblast proliferation
and as a weak chemoattractant (range 10−"$–10−"# M). At
this stage, a provisional matrix is deposited, which
attenuates fibroblast proliferation. The concentration of
TGF-β in the wound would then probably be much
higher owing to the stimulated increase in fibroblast
number. Other authors have suggested that at concentrations of above 10−* M, TGF-β is a potent stimulant
of collagen production [37]. Thus at approx. 10−* M,
TGF-β activity is adapted to collagen matrix deposition,
with stimulated functions of fibroblast-mediated contraction, secondary to stimulated fibroblast migration
and matrix remodelling. Hence, during the normal
evolution of the scarring process, the key functions
TGF-β in conjunctival scarring
of the fibroblast depend on its environment. In particular, the effects of growth factors determine fibroblast activity at any one time. Our in vitro results
thus suggest that the biphasic effects of TGF-β determine the response of fibroblasts, and that the role
of TGF-β during HTF-mediated conjunctival wound
healing are very much dependent on its concentration
at the wound site.
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 biological chorioretinal
‘ glue ’ for use in repairing retinal tears [38] and macular
holes [39]. Glaser et al. [39] suggested that visual acuity
following TGF-β2 treatment in macular hole surgery,
significantly improved in a dose-related manner (range
0.28–5.32i10−( M).
Early work by Roberts et al. [16] showed that a
subcutaneous injection of TGF-β1 and TGF-β2 (0–16i
10−( 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 [40], and application
of TGF-β2 on healing fractures in the rabbit suggested it
promoted callus formation in stable, but not unstable
conditions [41].
The effects of exogenous TGF-β application have also
been studied in several models of dermal wound healing.
Shah et al. [42] have demonstrated that exogenous
application of TGF-β1 to a linear incisional wound in rat
skin, affected the response in a dose-dependent manner.
Koch et al. [43] have shown that the effects of endogenous
TGF-β are different from the effects of supraphysiological exogenous doses, such that TGF-β1 null-mice did
not have impaired healing, although interestingly Smad3
(a key downstream mediator of TGF-β) null-mice,
showed accelerated healing of cutaneous incisional and
ionizing radiation wounds with reduced inflammation
and accumulation of matrix [44].
In one of the few studies that compared the in vivo
effects of all three TGF-β isoforms, Shah et al. [10]
applied exogenous TGF-β1, TGF-β2 and TGF-β3 to
this same incisional rat model of dermal scarring. They
suggested that TGF-β3 inhibited scarring and promoted
better collagen organization compared with TGF-β1 and
-β2, which stimulated dermal scarring. Subsequently,
however, Cox [17] demonstrated that TGF-β3 application, both in thermal wounds in mice in and incisional
and second intention wounds in rats, stimulates the
cutaneous scarring response in a manner similar to that
associated with TGF-β1 and TGF-β2. Our results suggest
that all three isoforms behave similarly in ocular scarring.
Important drawbacks in anti-scarring strategies that
are used at present for the eyes, such as the use of
MMC, are side-effects, associated complications, and
extensive microscopic and destructive cellular effects
[45–47]. This study demonstrates a new, more ‘ physiological ’ agent which effectively inhibits TGF-β2mediated conjunctival scarring activity in vitro and
appears clinically safe, non-toxic and well-tolerated, on
subconjunctival administration in vivo. It significantly
improves glaucoma filtration surgery outcome in an
animal model of aggressive conjunctival scarring.
Although agents that neutralize TGF-β have not
previously been used in the eye, they have been investigated in dermal scarring. By injecting anti-TGF-β1 and
anti-TGF-β2 (a polyclonal antibody raised in rabbit
against porcine platelet TGF-β2) antibodies, Shah et al.
[10] showed that they could significantly decrease the
scarring response to the same degree as the effects
produced by exogenous TGF-β3 in rat dermal excisional
wounds. These antibodies, however, have not been
studied clinically as they are unsuitable for human
use and, importantly, the role of TGF-β2-neutralizing
antibodies in post-surgical ocular scarring, specifically
following glaucoma filtration surgery, has not been determined. The novel technique used to produce the antiTGF-β2 monoclonal antibody used in this study, made it
different from other available anti-TGF-β monoclonal
antibodies, particularly because of its specificity for
human TGF-β2 and its high affinity for the active, rather
than the latent, form [48,49].
In summary, we have demonstrated that TGF-β is not
only a potent stimulant of HTF activity in vitro, but also
of the conjunctival scarring response in vivo. Since all
three isoforms are probably present during the woundhealing response after glaucoma filtration surgery, an
important finding has been the fact that they behave in a
similar manner. The actions of TGF-β are characterized
by different concentration-dependent effects and different peak activities for stimulating various fibroblast
functions, and its biphasic characteristics have implications for the timing and development of the conjunctival
scarring response. As TGF-β2 appears to be such an
important component of conjunctival scarring, it is not
surprising that its activity makes it a possible target for
modulating the scarring response following glaucoma
filtration surgery. We have shown that an anti-TGF-β2
antibody is effective in reducing subconjunctival scarring
following glaucoma filtration surgery in the rabbit, and
that a TGF-β antisense oligonucleotide can reduce
conjunctival scarring in a mouse model. The TGF-β2
antibody is now being assessed clinically for its efficacy
in improving glaucoma surgery results in patients.
Our studies show that TGF-β plays a major role in
conjunctival scarring, specifically following glaucoma
surgery. This is the first time such specific TGF-βtargeting agents have been used in filtration surgery, and
it represents an exciting new field for development in
inhibiting post-surgical scarring in the eye, without,
perhaps, the severe side effects associated with existing
modulating agents.
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M. F. Cordeiro
ACKNOWLEDGMENT
20
Financial support was by The Wellcome Trust Vision
Research Fellowship.
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