Effect of muscarinic agents on sclera fibroblast and their role in myopia 1

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I. Introduction

1.1 Myopia
Myopia has reached epidemic proportions in Singapore (Rajan et al 1994, Saw et al 1996).
Myopia is 1.5 to 2.5 times more prevalent in adult Chinese residing in Singapore than
similarly aged European-derived populations in the United States and Australia where the
sociodemographic associations are similar (Wong et al 2000). In the United States 25% of
children become myopic and it affects 15-20% of the adult population (Sperduto et al
1983, Hirsch and Weymouth 1990). Optically, myopia is defined as a mismatch between
the refractive optics and the length of the eye that causes an image to be focused in front of
the retina leading to an out of focus image. High myopia is an important cause of visual
disability (Klein et al 1995). It has been noted as the cause of blindness due to the many
associated complications, such as retinal break, retinal detachment and myopic retinopathy.

Myopia also places a burden on society and on the individual. The cost of glasses, contact
lenses and refractive surgery has been estimated to be $13 billion annually (Sheedy 1996).
Quality of life issues associated with myopia are also considerable. Myopia limits career
choices, social interaction and in underdeveloped countries, myopes may not have
corrective choices. Despite the high prevalence and associated social and economic costs
of correcting myopia, we know little about the etiology of myopia.




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1.1.1 Refractive development

Human studies have found that myopia results from axial elongation of the sclera that is not
corrected by a concommitant change in corneal curvature.
At birth, the cornea and lens are sharply curved so the focal plane is short. As the eye
matures, the axial length increases, rapidly at first during the “infant” high growth period
and then slowly during the “juvenile” slow elongation period (Sorsby et al 1961). Growth
moves the retina away from the cornea and toward the focal plane so that eventually the
axial length matches the focal plane, producing an emmetropic eye that focuses distant
objects without accommodation. In some cases, the axial length becomes longer than the
focal plane, so the image of distant object is focused in front of the retina causing blurring.

The eye is approximately 17mm long at birth. From birth to age 6, the eye grows by
approximately 5 mm. During this period there will be a loss of 4D of corneal power, and
20D of lens power. Through the process of emmetropisation, the distribution of refractive
error becomes narrow and the prevalence of myopia is only 2% at age 6. During the next 8
years, the average eye will grow approximately a millimeter. The prevalence of myopia
during this time will increase more than seven fold, to 15% by 15 years of age (Mutti et al
1996).

Gross and Wickman (1995) concluded that both emmetropisation and juvenile onset
myopia are best explained by a retinal image-mediated biochemical mechanism that
modulates eye growth. However, the nature of this growth mechanism has not been
elucidated.
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The emmetropisation mechanism in chickens and tree shrews appear to require visual
signals to guide the elongation of the eye. If the focal length is artificially lengthened by
wearing a minus-power lens, the eye will elongate until the axial length approximately
matches the amount of increase in imposed focal length (Irving et al 1991, Siegwart et al

1993). When the eyes are deprived of form vision with a translucent diffuser, there is no
visual image to indicate that the appropriate axial length has been reached. In this
situation, elongation continues unchecked, moving the retina past the focal plane (Pickett-
Seltner et al 1988, Wallman et al 1987).

If humans have an emmetropisation mechanism similar to that demonstrated in animals,
juvenile-onset myopia may occur if a child inherits a dysfunctional emmetropisation
mechanism. It is not clear if dysfunction occurs in photoreceptors, in the communication of
an unknown signal to the sclera, or in some intrinsic control of sclera growth. This
suggests that there may be an emmetropisation feedback loop, and this becomes disrupted
in myopia. Several studies have found pharmacological treatments that reduce axial
elongation in animal models (Stone et al 1989, McBrien et al 1993, Rohrer et al 1993,
Seltner et al 1993), and there are current clinical trials testing some of these in children.



1.1.2 Genetic influences
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Several lines of evidence point to a role of genetics in the development of myopia.
Monozygotic twins tend to resemble each other more closely in refractive error than do
dizygotic twins. Estimates of heritability (proportion of phenotypic variance explained by
heredity) for myopia obtained from monozygotic twins were higher than dizygotic twins
(Minkovitz et al 1993,Teikari et al 1988), suggesting a genetic influence.

A family history of myopia is associated with the likelihood of developing myopia, although
this could also be a result of visual habits such as amount of reading from parents. A
greater prevalence of myopia exists among the children of myopic parents than among the
children of non-myopic parents. According to the Orinda longitudinal study, prevalence of

myopia in children with two myopic parents is 30-40% whereas it is reduced to 20-25% in
children with one myopic parent and to <10% in children with non-myopic parents (Zadnik
et al 1994). Overall the parental history of myopia explains significantly more variance in
children’s refractive error and ocular component values than children’s near work activity
(reading, watching television, playing video games). It is unknown, however to what
extent these familial patterns are due to genetic or environmental factors. Children from
ages 6 to 14 years at risk for the onset of myopia due to a positive family history, have
longer eyes than children of similar age without myopic parents, even before the onset of
juvenile myopia (Zadnik et al 1993).
The association between increased axial length and juvenile onset myopia is known largely
from cross-sectional studies and from limited longitudinal investigations (Fledelius et al
1980, 1982, Sorsby et al 1970, Tokoro et al 1969). However, exactly how and why
normal eye growth goes awry to produce myopia is still unknown.
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1.1.3 Environmental influences
Throughout the world, myopia is becoming a greater problem in ocular health. A visual
feedback model of emmetropisation assumes that the eye modulates its growth in response
to visual input. It follows that refractive errors could arise from failures in either input or
the output from this feedback. Variation in the prevalence of myopia associated with
changes in near work demands also supports this view. It has been shown that myopia
increased in an urban Chinese population when compared to a group from a rural setting
(Lam et al 1995). The prevalence of myopia increased greatly in young persons compared
with older individuals among Alaskan Eskimos, Canadian Inuit, members of a Labrador
community, Yupik Eskimos, and American Indians in Ontario. The increase in myopia
incidence in Arctic regions coincided with the establishment of compulsory schooling
(Young et al, 1969, Johnson 1988, Johnson et al 1979, Alward et al 1985, Boniuk, 1973).

There is a much higher prevalence of myopia in Singapore. This has been attributed to its
rigorous education system and high near work demands (Au Eong et al 1993, Wong et al
2000). Other research points to the association of myopia and increased “near work” such
as reading during childhood (Angle et al 1980, Richler et al 1980). In addition,
experiments using animal models of myopia have demonstrated that an environmental
component has an important role in eye growth (Raviola et al 1985, Wallman et al 1987).

1.1.4 Experimental Myopia
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Animal models of myopia
Animal models have been developed to determine the influence of accommodation and the
visual environment on emmetropisation and refractive error. Two different types of
myopia have been developed. They are Form Deprivation Myopia (FDM) and Lens
Induced Myopia (LIM).
Animal studies on myopia began in the 1960’s (Young 1961, Lauber et al 1965), and
several animal models have been developed (Wiesel and Raviola 1977, Sherman et al 1977,
Wallman et al 1978, Somers 1978, Wilson and Sherman 1977). Several species have been
examined and three models have emerged such as chick (Wallman et al 1978), tree shrew
(Sherman et al 1977) and monkey (Wiesel and Raviola 1977).

Animal models have provided new insights into the understanding of myopia; they have
allowed studies into the cellular mechanism, which can not be undertaken in humans.
Whether these mechanisms are exactly similar to those controlling axial growths in humans
requires careful analysis as mentioned by Zadnik and Mutti (1995).
Animal experiments provide a basis for understanding the mechanisms that have evolved to
control the refractive development of the mammalian eye and it is probable that some of
them are common to all species.

1.1.5 Methods of inducing experimental myopia
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Form Deprivation Myopia (FDM)
This method for producing experimental myopia deprives a young eye of form-vision but
not light by means of lid-suture or the wearing of translucent diffusers. This form of
myopia can be produced in animal models such as chicks (Hodos et al 1984, Sivak et al
1990, Wallman et al 1987,1978), monkeys (Tigges et al 1990, Wiesel et al 1977),
marmosets (Troilo et al 1993), tree shrews (Marsh-Tootle et al 1989, McBrien et al 1992),
guinea pigs (Lodge et al 1994), squirrels (McBrien et al 1993) and rabbits (Beuerman et al
1993).
Lid suture was used in young monkeys to produce myopia (Wiesel and Raviola, 1977).
After suturing the lids for periods up to 26 months the eyes were found to be myopic by
retinoscopy (-1 to -13.5D). Both axial length and equatorial diameter were increased. The
posterior sclera was thinner than the fellow control eye while the anterior segment of the
eye was similar on the control and experimental two sides. Wiesel and Raviola carried out
further experiments and found that lid-suture in dark reared animals with lid-suture did not
alter the refraction or axial length (Raviola et al 1978). They also reported in 1979 that
opacification of cornea by injection of latex particles produced an increase in axial length in
primates (Wiesel et al 1979). This result can also be explained as the effect of a reduction
in pattern vision. Raviola and Wiesel (1985) went on to examine the effects of optic nerve
section on lid suture myopia. In one stump tailed macaque myopia was prevented by optic
nerve section, while in three rhesus macaques it was not. This suggested that in the retina
might release regulatory molecules whose production is influenced by the pattern of light
stimulation.
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In newly hatched chicks myopia can be produced either rapidly by lid suture or wearing
simple occluders. The effects of lid-suture on the morphology and refractive state of the
chick eye have been extensively described (Yinon 1980,1983). The morphological changes
included increase in axial length and equatorial diameter. It has also been reported that
dark rearing results in the enlargement of the posterior segment, accompanied by corneal
flattening resulting in a net hyperopia when combined with lid suture and light diffusers
(Yinon 1986, Gottleib 1987). It should be noted that dark rearing does not only remove
the visual input but it changes the circadian rhythms.

Lens Induced Myopia (LIM)
The other form of experimental myopia, lens induced myopia results from raising an animal
with a negative lens over one eye, imposing hyperopic defocus (image behind retina)
leading to axial elongation. In a similar manner, hyperopia can be induced by raising
animals with positive spectacle lens over the eye to impose myopic defocus (image focused
in front of the retina).
Lens induced myopia and hyperopia were developed first in chickens and more recently in
rhesus monkeys, tree shrews, and marmosets (Schaeffel et al 1988, Irving et al 1991,1995,
Siegwart and Norton 1993, Hung et al 1995, Graham and Judge 1995,1999).
This important observation suggests that the eye can actively control growth to optimise
visual acuity. This finding could be a clinical problem because negative lenses are fitted to
children with growing eyes to correct refractive error. It is therefore important to
understand this growth control mechanism, and whether it occurs in humans.
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To follow up on the finding that the application of a simple lens to the eye of the young
chick can produce an eye enlargement, experiments have been designed to occlude the
whole visual field as well as a portion of the visual field. Domes, arches and rings, were

applied to the eye of three-day old chicks (Hayes et al 1986). The domes degraded the
image over the entire visual field of one eye. The arches degraded only the lateral visual
field, leaving unobstructed vision in the frontal and posterior visual field. The rings did not
occlude the visual field, but served as a control. Chicks were refracted in the lateral visual
field at the age of 3 to 7 weeks. The application of the dome device in particular produced
a large shift to myopic refraction. The rings did not affect eye growth while the arch
significantly increased the dorsoventral equatorial diameter of the eye. The dome device
had the most dramatic effect on eye morphology and resulted in an increase in both axial
and equatorial dimensions. Eyes fitted with the dome device had a steepened cornea,
increased anterior chamber depth, more open angle and a greater corneal diameter than
control eyes. The axial length of the posterior segment was also increased. These results
suggested scleral growth be topographically related to the sector of the retina undergoing
visual deprivation (Hodos et al 1984, 1985, Hayes et al 1986).

In the chick, experimental myopia developed even after optic nerve section. Thus, the
overgrowth is produced by systems confined to the eye and need not involve visual
feedback from the brain to the eye (via accommodation or some other efferent system). An
explanation could be that retinal cells sensitive to image quality could produce substances,
which locally modulate sclera growth. However, there is no mechanism to explain that
effect.
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1.1.6 Recovery from experimental myopia
Wallman and Adams (1987) investigated the susceptibility to and recovery from axial
myopia in the chick produced by occluders. Recovery occurred when the occluders were
removed within the first six weeks and amounted to 20 D in two weeks. Troilo and
Wallman (1988) have reported that chick eyes made hypermetropic by dark rearing or
myopic by occluders can recover toward emmetropia when reverting to normal visual

conditions. Recovery was also found to proceed when the Edinger Westphal nucleus was
lesioned (eliminating accommodation) and when the optic nerve is cut.

Chicks were found to compensate for a large range of imposed refractive errors. There
was nearly complete compensation within a week for lenses from -10 to + 20 D (Irving et
al 1992), with greater compensation for positive lenses than negative lenses. Whether this
bi-directional compensation occurs in humans is uncertain.

1.1.7 Clinical models of experimental myopia
Clinical observations have suggested that a condition of image deprivation myopia occur in
humans that are similar to FDM in animal models.
Birth injuries of the cornea, often unilateral have been associated with subsequent myopia
(Lloyd 1938). Myopia has been observed in patients with corneal opacities (Muramatsu
1982). Premature infants with or without retinopathy of prematurity (ROP) may develop
abnormal eyes with impaired vision, refractive errors and strabismus. Numerous studies on
refraction in pre-term infants have shown a predisposition to childhood myopia ( Birge
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1956, Kalina 1969, Fledelius 1976, Shapiro et al 1980, Kushner 1982, Koole et al 1990,
Gallo et al 1991, Fledelius 1993).
Johnson and colleagues (1982) have described axial myopia in one eye in one sibling of a
pair of identical twins, which had a posterior subcapsular cataract.
Von Noorden and Lewis (1987) examined 10 young patients who had unilateral cataracts
and in seven out of ten cases the involved eye revealed increased axial length.
Robb (1977) described an association between hemangiomas of the eye and high myopia in
young children. Hoyt and colleagues (1981) described eight infants with lid closure caused
by nerve palsy, obstetric trauma, and hemangioma, all were found to have increased axial
length and myopia.

1.1.8 Species differences
Few experiments have been carried out on compensational myopia in mammals. A main
difference between birds and mammals is the time course of the response to visual
deprivation and the structure of the sclera.

In chicks, when deprivation is removed, the rate of ocular elongation returns to normal
within days. In contrast, monkey eyes continue to elongate for months after visual
deprivation is removed. If marmosets are visually deprived during the period of normal
emmetropisation and form deprivation ends, the eye continues to elongate and become
increasingly myopic for several months (Troilo et al 1993).

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Mammalian models of myopia such as tree shrews and monkeys have an advantage over
avian models as the structure and biochemistry of their eyes is more similar to humans.
Sherman, Norton and Casagrande (1977) introduced tree shrews as a model for myopia.
Visually induced changes in eye growth of tree shrew and monkey are smaller than that of
chickens and continuous treatment with diffuser spectacle or lid suture is more difficult but
has been successful (Wiesel and Raviola 1977,1985, Smith and Hung 1999,2002, Smith et
al 2000).
The major obstacle in using monkeys are the longer time required for the effect and
additional level of care. There are many results from chickens that have not yet been
obtained from monkeys but are essential for the understanding of human myopia.
The chicken is currently the most frequently studied animal model due to its easy
availability and advantages of rapid eye growth, good optical quality and sensitivity to
moderate modifications of vision. The chick can develop up to 20D of axial myopia in a
week and can compensate for imposed defocus of 4D in 3 days. The chicken model was
introduced by Wallman, Trachtman and Turkel (1978).
However the results from chickens are difficult to apply to mammals. The pharmacology

and morphology of pupillary and accommodation responses are different in chicks and
mammals. Fortunately, there are similarities with regard to the roles of neurotransmitters
and neuropeptides, such as acetylcholine, dopamine and vasoactive intestinal polypeptide
(VIP) in the control of eye growth (Schaeffel et al 1995, Stone et al 1988).



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1.1.9 Pharmacology of myopia
Several studies have reported the modulation of the effect of pharmacological agents on
experimental myopia. Raviola and Wiesel (1985) found that atropine had no effect in the
rhesus macaque, but prevented axial elongation in the stump-tailed macaque. Wildsoet and
Pettigrew (1988) have shown that intravitreal injections of neurotoxin kainic acid induced
axial elongation in chickens. Kainic acid is a glutamate analogue, while another glutamate
analogue amino-phosphonobutyric acid also (APB) has been reported to reduce axial
elongation (Smith, Fox and Duncan, 1985).
It was observed that retinal concentrations of neurochemicals were altered in experimental
myopia, specifically VIP was increased in monkey and dopamine was decreased in both
chick and monkey (Iuvone et al 1989, Stone et al 1988,1989).
A recent study found that glucagon-containing amacrine cells respond differentially to the
sign of defocus and may mediate lens-induced changes in ocular growth refraction (Fischer
et al, 1999). Local application of the dopamine agonist, apomorphin blocked the axial
elongation that ordinarily follows visual deprivation in chick and in rhesus monkey (Stone
et al 1989,1991, Iuvone 1991). In chicks, normalisation of retinal dopamine correlated
with recovery from myopia (Pendrak et al 1997).
Use of atropine
Historically atropine was used because it is a cycloplegic agent in human antagonising the
mAChRs of the cilliary muscle. It also reduces axial elongation in deprivation models of

myopia (McBrien et al 1993, Stone et al 1991).
In the case of chicks, the ciliary muscle is striated and therefore is innervated via nicotinic
rather than muscarinic cholinergic receptors (McBrien et al 1993). Therefore the effect of
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atropine must have been exerted on the retina or directly on the sclera. Indeed, there is
evidence that atropine inhibits cellular proliferation of sclera chondrocytes and extra
cellular matrix production (Marzani et al 1994, Lind et al 1997).

Atropine has been advocated as a myopia therapy for some time, based on the assumption
that it prevents the accommodative response. Several studies have shown that daily topical
ocular instillation of 1% or 0.5% atropine markedly reduces the progression rate of myopia
(Sampson 1979, Bedrossian 1971, Brodstein et al 1984, Dyer 1979, Goss 1982, Brenner
1985, Kao et al 1988, Kelly et al 1975, Yen et al 1989, Shih et al 1999). Side effects,
systemic and ocular such as blurred vision, photophobia, allergic dermatitis, possible UV
related retinal damage and direct effect of the heart rate make long term use of atropine
impractical (Brodstein 1984, Chou et al 1997).

Muscarinic receptors (mAChRs)
Muscarinic receptors are members of a superfamily of G protein coupled receptors.
Molecular cloning studies identified five subtypes of mAchR genes (m1-m5) widely
expressed in various tissues (Kubo et al 1986, Peralta et al 1987, Hulme et al 1990,
Receptor Nomenclature suppl, 1993). All these receptors share a similar three-dimensional
structure consisting of a tightly packed bundle of seven transmembrane helices linked by
three extracellular and three intracellular loops (Wess et al 1995). Pharmacological studies
have revealed four different subtypes of mAChR (m1-m4) based on their ability to bind
specific ligands and activate second transduction pathways. Intracellular signals are
transmitted by coupling of receptors to cytoplasmic guanine nucleotide-binding regulatory
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proteins (G proteins) (Hulme et al 1990, Lanbert et al 1992) with subsequent generation of
second messengers.
The molecular weight of the subtypes predicted from amino acid sequences range between
51 kd and 66 kd. (Kerlavage et al 1987, Ashkenazi et al 1988).

1.2 Sclera
Alteration in visual experiences can increase scleral growth, leading to axial elongation and
myopia (Hodos et al 1984, March-Tootle et al 1989, Sherman et al 1977, Tigges et al
1990, Wallman et al 1978, Wiesel et al 1977, Yinon et al 1980). It has been reported that
the cartilaginous part of the sclera of form deprived chick eyes is 21% thicker and has more
dividing chondrocytes (Christensen et al 1991). Increased proteoglycan synthesis has also
been reported in myopic eyes (Rada et al 1990). Furthermore, sclera of myopic chick eyes
was reported to have increased latent metalloproteinases (MMPs) suggesting that myopia
may be a result of sclera remodelling during growth.

A mechanical hypothesis has been suggested to explain excessive axial elongation. It has
been suggested that myopic eyes may experience higher externally applied forces, such as
from blinking or rubbing of the eye (Greene 1979, Ku and Green 1981), higher intraocular
pressure (Arciniesgas 1980, Maurice and Mushin 1966, Tokoro 1970, Pruett 1988) or
higher extra ocular muscle tensions (Greene 1980). It has been suggested that these higher
forces may result in sclera creep.
Another possible cause would be the elastic stretching of the sclera shell due to lower
elastic modulus in the myopic eye. Tree shrew sclera from myopic eyes does appear to
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stretch more easily than control eyes. Philips and McBrien (1995) studied this in tree
shrews by measuring the modulus of elasticity and sclera thickness of tree shrew eyes that

were monocularly form-deprived. They simulated the elastic deformations of the control
and form-deprived eyes with a finite element model and found that both the measured
sclera thickness and elastic modulus were lower in the myopic eyes. Weakening of the
sclera in high myopia may be the result of the structural change (Curtin et al,1958).
Biochemical and structural studies of myopic eyes support the idea that sclera growth and
remodelling both contribute to the development of myopia.
For example, lower levels of extracellular matrix proteins and collagen content have been
observed in the sclera of experimentally myopic tree shrew (Norton et al 1995). This could
be due to increase activity of matrix metalloproteinases (MMPs), decreased activity of
tissue inhibitors of metalloproteinases (TIMPs) or a fundamental lack of protein synthesis.
Similarly, light and electron microscopic analyses of myopic monkey and human eyes have
demonstrated changes in collagen fibre diameter and bundle spacing (Funata 1990, Liu
1986).
All these studies suggest that scleral elongation in experimental myopia involves more than
just passive stretching of the sclera and biochemical or molecular changes are probably
associated with growth and remodelling.
The cardinal characteristic of myopia is axial elongation of the posterior part of the eye.
All animal models have developed excessive vitreous chamber elongation (Wallman et al
1978, Norton et al 1990). Active sclera growth is a primary event in the axial elongation
therefore control of the sclera fibroblast may lead to a possible therapy for myopia
prevention or decreasing the development of myopia.
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1.2.1 Matrix Metalloproteinases (MMPs)
Evidence has been obtained that the sclera extracellular matrix (ECM) remodelling of the
sclera is part of myopic process. Increased levels of gelatinase A was observed in the
sclera of experimentally myopic chick eyes (Rada et al 1995,1999).

MMPs are a family of related zinc metalloproteinases that are involved in normal
development, wound healing, cancer, arthritis, and angiogenesis. MMPs are secreted as
inactive pro-enzymes. Activation of these zymogens is the critical step that leads to tissue
catabolism. MMP pro-enzymes are activated by proteinases, thiol modifying reagents
(mercurials, oxidised glutathion, HOCl ) and heat. The endogenous protein inhibitors
(TIMPs) inhibit activated MMPs. There are at least 17 different MMPs and 4 TIMPs
expressed in human tissue (Woessner 1998). The balance of between MMP and TIMP
expression determines ECM homeostasis.

Growth factors and hormones can regulate MMP and TIMP expression. Various cell types
including fibroblasts produce TIMPs. Their expression is regulated by various cytokines
such as Interlukin-1,6,11 (IL-1, IL-6, IL-11), transforming growth factor (TGF-β) and
epidermal growth factor (EGF) (Roeb et al 1993, Leco et al 1994,1993). Selective
stimulation of MMP is under control of growth factors such as fibroblast growth factor
(FGF), vascular endothelial growth factor (VEGF) and tumour necrosis factor (TNF-α)
(Moses 1997, Wojtowicz-Praga et al 1997).

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1.2.2 Sclera fibroblast control
Sclera cells are the final effectors in a complex signal cascade leading to axial elongation.
Several studies have established the effectiveness of atropine in decreasing the progression
of axial length in humans and in experimental myopia, however the mechanism is still
elusive. Atropine may act directly on sclera fibroblasts through the G protein coupled
muscarinic receptor to modulate the development of myopia.
It has been observed in myopic eyes that some growth factor expression levels were
altered. FGF-2 level was significantly lower in the sclera of myopic eyes than the control
and TGF-β level was significantly higher in myopic eyes (Seko et al 1994). Atropine may
have an effect on growth factors, which then control sclera fibroblast activity through

tyrosine kinases, MMPs and TIMPs.

1.3 Growth factors in myopia
Intravitreal and subconjunctival injections of FGF-2 have prevented axial elongation
induced by form deprivation (Rohrer et al 1993,1994). FGF-2, which is reduced in myopic
eye, may provide a “stop” signal for the growing eye although it is generally regarded as
fibroblast proliferative factor. The effect could be mimicked by FGF-1 but with potency
approximately 160 times less than that of FGF-1.
TGF-β was not found to induce myopia or to increase myopia however it acted as a potent
inhibitor of FGF-1 when injected together with FGF-1 (Rohrer et al 1994)

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Phosphorylation of tyrosine kinases receptor
Tyrosine kinases couple cell surface events to the regulation of many intracellular activities
such as gene expression, proliferation and ion channel modulation. Studies show that
growth factors, cytokines, integrins, antigens and G protein coupled receptors also utilise
tyrosine kinase pathways to transduce intracellular signals (Zachary and Rozengurt 1992,
Aoki et al 1994, August et al 1994, Chen et al 1994, Ihle et al 1994).

1.3.1 Fibroblast Growth Factor (FGF)
The FGF family has at least 18 different isoforms, which display a remarkable affinity to
heparin and are also called heparin binding growth factors (HBGF).
FGF-2 is an 18Kd protein consisting of 155 amino acids, and an isoelectric point of 9.6,
without disulfide bonds or glycosylation. Some higher weight forms of FGF-2 (22-24Kd)
have been also described. The smallest form (18Kd) occurs predominantly in the cytosol
while the higher molecular weight forms are associated with the nucleus and ribosomes.
An isoform of FGF-2 associated with the endoplasmic reticulum has been designated
altFGF-2 (Burgess and Maciag 1989, Baird and Klagsburn 1991, Goldfarb 1990,

Gospodarowicz et al 1987).
FGF-2 is found in almost all tissues of mesodermal and neuroectodermal origin and also in
tumors derived from these tissues. Endothelial cells produce large amounts of FGF. Some
FGF-2 is associated with the extracellular matrix of epithelial and endothelial cells
(Abraham et al 1986). Many cells express FGF-2 only transiently and store it within the
cytoplasm in a biologically inactive form (Bugler et al 1991).
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FGF-2 is released after tissue injury (Fiddes et al 1991, Hebda et al 1990), during
inflammatory processes and also during the proliferation of tumor cells (Chodak et al 1988,
Fujimoto et al 1991).
At the sequence level, FGF-1 and FGF-2 display 55 percent homology. FGF-1, like FGF-2
does not possess a signal sequence that would allow secretion of the factor by a classical
secretion pathway. The mechanism underlying the release of FGF-2 is unknown (Baird and
Klagsbrun 1991, Burgess and Mariag 1989, Goldfarb 1990).

A truncated variant of FGF-1 generated by alternative splicing and removal of the entire
second coding exon is only 60 amino acids long (6.7 Kd) elicits only minimal fibroblast
proliferation and antagonises the effect of full length FGF-1 when added exogenously or
when co-expressed with FGF-1 in 3T3 fibroblasts (Yu et al 1992). FGF-1 binds to the
same receptor as FGF-2. FGF-1 displays similar activities as FGF-2 however, it is 50-100
fold less active than FGF-2.

Fibroblast growth factor receptor (FGF-R)

FGF receptors are encoded by a gene family consisting of at least four receptor tyrosine
kinases (FGF-R1,2,3,4) that transduce signals for cell growth and differentiation
(Armstrong et al 1992, Avivi et al 1993, Eisemann et al 1991, Keegan et al 1991). Binding
of FGF-2 to one of its receptors requires the interaction with heparan sulfate and heparan

sulfate proteoglycans (syndecan) of the extracellular matrix (Keifer et al 1991, Ornitz et al
1992, Raprager et al 1991). Heparan has been shown to protect FGF-2 from inactivation
by proteases, acids and heat. Heparan also increases the biological activity of FGF-2.
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Binding of FGF-2 (also FGF-1) to receptor can be inhibited by suramin and also by
protamin (Yayon et al 1991).
It has been observed that binding of FGF-R leads to phosphorylation by an intracellular
protein kinase, which may additionally alter FGF activity and bioavailability (Partanen et al
1992, Vainikka et al 1992, Werner et al 1992, Moscatelli 1991).


Biological activity
A multifunctional role of FGF-2 is suggested by the many different receptor phenotypes
and their cell specificity (Alarid et al 1991, Gallicchio et al 1991). FGF-2 stimulates the
growth of fibroblasts, myoblasts, osteoblasts, neuronal cells, endothelial cells,
keratinocytes, chondrocytes and many other cell types (Araujo and Cotman 1992, Benezra
et al 1992, Davidson and Broadley 1991, Ray et al 1993, Sato et al 1991). FGF-2 has also
been shown to be a promoting or inhibitory modulator of cellular differentiation. In early
embryos FGF-2 functions as a differentiation factor that induces tissue destined to produce
ectodermal structure to differentiate into mesodermal tissues. FGF-2 retards senescence of
cells thus allowing maintenance of cells in vitro which would normally lose their
differentiated phenotype in long-term cultures. An FGF-2 influence the differentiation
processes is probably the result of a complex interaction with other factors.
Transgenic animal studies show that FGF-2 is essential for the morphogenesis of suprabasal
keratinocytes and for the establishment of the normal program of keratinocyte
differentiation (Werner et al 1993).
22

Treatment of various human tumor cell lines (melanomas, glioblastomas) with antisense
RNA directed against FGF-2 have been shown to inhibit the proliferation of these cells and
their ability to form colonies (Becker et al 1989, Morrison 1991).

1.3.2 Transforming growth factor (TGF-β)
TGF-βs are secreted from cells as latent complexes. They are synthesised by macrophages,
lymphocytes, endothelial cells, keratinocytes, granulosacells, chondrocytes, glioblastoma
cells and leukaemia cells (Barnard et al 1990, Bonewald and Mundy 1989, Sporn and
Roberts 1992). TGF-β can be induced by steroids, retinoids, epidermal growth factor
(EGF), NGF, vitamin D
3
and Interleukin (IL-1). The synthesis of TGF-β can be inhibited
by EGF, FGF, retinoids, calcium and follicle stimulating hormone (Border and Ruoslahti et
al 1992, Burt 1992, Hooper 1991). TGF-β is stored in an inactive form as associated with
ECM and complexed with betaglycan and decorin. The mechanisms underlying its release
from these reservoirs is unknown. TGF-β exists in at least 5 isoforms. The amino acid
sequence of the various isoform display 70-80 percent homology. TGF-β
1
is the prevalent
form. TGF-β is the most potent known growth inhibitor for normal and transformed
epithelial cells, endothelial cells, fibroblasts, neuronal cells, lymphoid cells and other
hematopoietic cell types, hepatocytes and keratinocytes (Miyazono and Heldin 1992,
Pfeilschifter 1990, Robers and Sporn 1990, 1992,1993).

In many cell types TGF-β antagonises the biological activities of growth factors which have
receptors of the tyrosine kinase group. At extremely low concentrations, TGF-β is a
growth inhibitor for smooth muscle cells, fibroblasts and chondrocytes. At higher
23

concentration TGF-β stimulates proliferation of these cells. This bimodal activity is
mediated in part by platelet derived growth factor (PDGF). The synthesis and secretion of
PDGF is stimulated by low concentrations of TGF-β, while higher concentrations lower the
expression of PDGF receptors and hence diminish the biological effects of PDGF. TGF-β
stimulates the synthesis of ECM including collagen, proteoglycan, fibronectin and integrin
(Roberts et al 1990, Ignotz and Massague 1986).

EGF-R
Epidermal growth factor receptor (EGF-R) is a 170kd membrane bound glycoprotein
expressed on the surface of epithelial cells. EGF-R is a receptor tyrosine kinase known to
regulate cell cycle (Carpenter and Cohen 1987, Yarden and Ullrich 1988, Cadena and Gill
1992). The receptor is activated when its ligand (EGF, TGF-α) binds to the extracellular
domain, resulting in autophosphorylation of the receptor’s intracellular tyrosine kinase
domain (Cohen et al 1980, Schreiber et al 1983). Dimerisation and internalisation of EGF-
R molecules transduce proliferative signals. Several reports have indicated that EGF-R is
over-expressed in various cancers (Gullick et al 1986, Ozanne et al 1986, McKenzie et al
1991). There are also studies, which suggest a relationship between levels of EGF-R and
levels of estrogens and progesterone receptors (Klijin et al 1992, Nicholson et al 1991,
Sainsbury et al 1987, Koenders et al 1991).



1.4 Aims of Project
24


Atropine has been shown to prevent the development of myopia. Evidence is available to
show that atropine and more specific m1 muscarinic receptor antagonists, such as
pirenzepine, may slow axial elongation in both experimental and clinical myopia (Cottrial et

al 1996). Pharmacological treatment of myopia and its potential widespread application
raise a number of questions.
Experimental myopia leads to an increase in ECM production and accumulation of
proteoglycans within the cartilaginous sclera while fibrous sclera was opposite as similarly
observed in monkey and tree shrew. It has been suggested that increased growth of the
cartilaginous sclera is associated with interaction between cartilaginous sclera and fibrous
sclera (Marzani and Wallman 1997).
It is the hypothesis of this study that sclera fibroblasts may play a major role in axial
elongation and atropine has a direct effect on this cell to decrease myopic progression. The
general goal of this study has been to determine the cellular response of sclera fibroblasts to
atropine. A culture system for sclera fibroblasts will be developed and used to test the
effect of atropine on proliferation, receptor activity and intracellular signal transduction
pathways.
To test the overall hypothesis, the following specific phenomena will be studied.
1. The presence of muscarinic receptors in sclera fibroblasts (SF).
2. The response of SF to muscarinic agents.
3. The response of SF to growth factors.
4. The changes in growth factors and matrix metalloproteinases in response to atropine.
5. The changes in matrix metalloproteinases in response to growth factors
25


II. Materials and Methods

2.1 Materials
2.1.1 Animals
Female Lohman Brown chicks were purchased from a local hatchery (Chew’s agriculture,
Singapore) on the first day of hatching. They were reared in our animal facility on a 12:12
hour light:dark cycle (average 110 lux).
New Zealand White rabbits (6 weeks old) and Balb/c mouse (8 weeks old) were purchased

from the Animal Center of the National University of Singapore.

2.1.2 Reagents
Coomassie plus protein assay reagent (Pierce), Bovine serum albumin;BSA standard
(Pierce), M-Per mammalian protein extraction reagent (Pierce), Atropine sulfate( Sigma),
Pirenzepine (Sigma), Carbacol (Sigma), cyclic AMP (Sigma), ATP (Sigma), FGF-2
recombinant human protein (Oncogene), Anti muscarinic receptor (m1,m2,m3,m4,m5)
antibody (Biogenesis), Anti FGF-2 mouse IgG peroxidase conjugate, Human Proenzyme
MMP-2 proteins(Oncogene), Polyclonal anti MMP-2 (Calbiochem), Mouse monoclonal
anti MMP-2 antibody (Neomarkers), Anti-Sheep IgG peroxidase conjugate (Pierce), Anti-
mouse IgG peroxidase conjugate (Pierce), Anti rabbit IgG peroxidase conjugate (Pierce),
Bromodeoxyuridine;BrdU (Calbiochem), Anti BRDU antibody (Phrarmingen), DEVD-
pNA substrate (Promega), Anti FGF-1 rabbit IgG (Sigma), FGF-1 standard (Sigma), Anti
human TIMP mouse IgG (Calbiochem), DMEM (Sigma), Antibiotic-antimycotic (Sigma),
FBS (Hyclone), Sample buffer (0.5M Tris-HCl, Glycerol, 10% SDS, β-mercaptoethanol,