\set{final}

\def\Author{Frost}
\def\author{frost}
\def\vol{13}
\def\year{2007}
\def\anum{176}
\def\pages{1580-1588}
\def\txt_title{Differential protein expression in tree shrew sclera during development of lens-induced myopia and recovery}
\def\txt_authors{Michael R. Frost, Thomas T. Norton}

\def\rcvd{18 June 2007}
\def\accept{28 August 2007}
\def\publ{6 September 2007}
\def\pdfsize{}
\def\GalleyQCount{1}
\def\PMID{}


\include{mvstyle.hsm}

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\| Internal defs


\article{

\title{Differential protein expression in tree shrew sclera during
development of lens-induced myopia and recovery}

\authors{\mailto{mrf@uab.edu}{Michael R. Frost},
\mailto{tnorton@uab.edu}{Thomas T. Norton}}

\institutions{Department of Vision Sciences, School of Optometry,
University of Alabama at Birmingham, Birmingham, AL}

\correspondence{Michael R. Frost, PhD., 302 Worrell Building, Department
of Vision Sciences, University of Alabama at Birmingham, 924 18th Street
South, Birmingham, AL, 35294-4390; Phone: (205) 934 6733; FAX: (205) 934
5725; email: mrf@uab.edu}

\abstract

\abs_purpose{The tree shrew model of refractive development is
particularly useful because, like humans, tree shrews have a fibrous
sclera. Selective changes in some candidate extracellular matrix
proteins and mRNAs have been found in the sclera during the development
of and recovery from induced myopia. We undertook a more neutral
proteomic analysis using two-dimensional gel electrophoresis and mass
spectrometry to identify scleral proteins that are differentially
expressed during the development of and recovery from lens-induced
myopia.}

\abs_methods{Five tree shrews (\i{Tupaia glis belangeri}) wore a
monocular -5 D lens for four days, starting 24 days after natural eye
opening. At the end of this time, all treated eyes had partially
compensated for the lens and were -3.5\pom 0.7 D (mean\pom SEM) myopic
relative to the untreated fellow control eyes. An additional five
animals wore a -5 D lens for 11-13 days followed by four days of
recovery without the -5 D lens. The amount of recovery was 1.6\pom 0.4
D. Scleral proteins from both groups were then isolated and resolved by
two-dimensional gel electrophoresis and spots that were differentially
expressed were identified by mass spectrometry.}

\abs_results{The scleral protein profile typically displayed about 700
distinct protein spots within the pH 5-8 range. Comparison of the
treated-eye and control-eye scleras of the lens-compensation animals
revealed five spots that were significantly and differentially expressed
in all five pairs of eyes; all were downregulated 1.2 to 1.7 fold in the
treated eye. These proteins were identified as: pigment
epithelium-derived factor (PEDF), procollagen I\alpha 1, procollagen
I\alpha 2, and thrombospondin I (two spots). In the recovering eyes, the
two thrombospondin I spots remained lower in abundance while PEDF and
the procollagens were no longer downregulated. In addition, 78 kDa
glucose-regulated protein (GRP 78), a member of the heat shock protein
70 family, was slightly upregulated 1.3 fold.}

\abs_conclusions{We found consistent results across animals that were of
a magnitude consistent with the physiologically small changes to the
focal plane of these eyes. Changes in collagen confirm previous
findings, but downregulation of thrombospondin I adds detail to our
understanding of the chain of signals that regulates scleral creep rate.
The differential changes in PEDF and GRP 78 were not expected based on
previous studies and demonstrate the utility of the proteomic approach
in tree shrew sclera.}

\introduction

\p{Tree shrews are small mammals that are closely related to the primate
line [1,2] and have excellent vision for their size [3]. During normal
postnatal development, tree shrew eyes demonstrate visually-guided
emmetropization: visual signals regulate the axial elongation rate of
the growing eye so that the retina comes to reside at the focal plane,
producing an eye that is in good focus [4-6]. When a concave
(negative-power) lens is placed before one eye of a juvenile tree shrew
and held in a goggle frame [7], the eye is made artificially hyperopic.
In response, the eye rapidly elongates until, after 10-15 days, it has
compensated for the lens so that the retina again lies at the focal
plane and the eye is again emmetropic while wearing the lens [6,8-10].
If the lens is then removed, the eye is myopic. In juvenile animals,
"recovery" from the induced myopia occurs [11]. The axial elongation
rate of the eye slows dramatically while the optics of the eye continue
to mature until the retina is once again located at the focal plane.
Recovery occurs in most but not all juvenile tree shrews.}

\p{The sclera is the outer coating of the eye that, in addition to
protecting the retina and allowing the attachment of the extraocular
muscles, controls the size of the eye and the location of the retina
relative to the focal plane. Tree shrews, like primates, have a fibrous
sclera comprised largely of type I collagen along with lower amounts of
type III and type V collagen, elastin, proteoglycans, and other
structural proteins [12-18]. This extracellular matrix (ECM) is produced
by scleral fibroblasts and is arranged in interwoven layers or lamellae.
Both negative-lens compensation and recovery from induced myopia involve
changes in a biomechanical property of the sclera, viscoelasticity,
which is measured as creep rate, the rate of increase in the length of a
strip of sclera while under constant tension. The creep rate increases
during the early phase of negative-lens compensation, reaching a peak
after four days of lens wear, then declines toward normal as the eye
completes its compensation [8]. During recovery, creep rate falls
rapidly (within two days) to below normal values [8]. Underlying the
biomechanical changes is selective tissue remodeling that involves
alteration to both the synthesis and degradation of ECM components such
as collagen, proteoglycans, and glycosaminoglycans [12,19,20]. It
appears that these changes allow normal intraocular pressure to expand
the globe during negative-lens compensation, perhaps by increasing the
ease with which the scleral lamellae slip across each other.}

\p{Several studies have examined mRNA levels in sclera during
negative-lens compensation and recovery and have found selective
regulation for "candidate" proteins in tree shrews and chicks [21-24]
with a few of these changes also being detected at the protein level
such as collagen I, matrix metalloproteinase 2 (MMP-2), and TGF-\beta\
[12,19,20]. However, it is still largely unknown how most of the
observed changes in mRNA levels correlate to changes in protein levels;
protein levels that more accurately represent the state of a biological
system.}

\p{To complement our previous "candidate" approach that examined levels
of mRNA for specific targets, we have now undertaken a more neutral
proteomic analysis using two-dimensional gel electrophoresis (2DGE) and
mass spectrometry which has the potential to identify any additional
scleral proteins that are differentially expressed during myopia
development and recovery. In 2DGE, proteins extracted from the sclera
are first separated by pH using isoelectric focusing and are then
separated by molecular weight using a standard SDS-PAGE gel, producing a
unique pattern of protein spots. Comparison of gels from treated and
control eyes reveals changes in the abundance of individual proteins
that are subsequently collected from the gel and identified using mass
spectrometry.}

\methods

\subsection{Experimental groups}

\p{Juvenile tree shrews (\i{Tupaia glis belangeri}) were produced in our
breeding colony and raised by their mothers on a 14 h on/10 h off
light/dark cycle. All procedures complied with the ARVO Statement for
the Use of Animals in Ophthalmic and Visual Research and were approved
by the Institutional Animal Care and Use Committee of the University of
Alabama at Birmingham.}

\p{Groups of tree shrews (n=5 per group), balanced to include both males
and females and avoiding pups from the same parents within a group, were
divided into two conditions: a negative-lens compensation group and a
recovery group. As in previous studies [8,23], the negative-lens
compensation group wore a monocular -5 D lens (spherical power) for four
days starting 24\pom 1 days after natural eye opening (days of visual
experience [VE]) to induce axial elongation and myopia. The recovery
group experienced -5 D lens wear for 11-13 days also starting at 24\pom
1 days of VE to induce negative-lens compensation. Then the lens was
removed and the now-myopic treated eye was allowed to recover for four
days. These lens-wear periods were chosen because mRNA studies had
previously shown significant expression changes at these time-points
[21,23].}

\subsection{Pedestal attachment}

\p{To attach the goggle containing the -5 D lens firmly to the head
during lens treatment, at 21\pom 1 days of VE, all animals were
anesthetized (17.5 mg ketamine, 1.2 mg xylazine, supplemented with
0.5-2.0% isoflurane as needed) and received a dental acrylic pedestal
following procedures described by Siegwart and Norton [7]. Three days
later, the goggle frame was clipped to the pedestal. Animals in both
groups wore a monocular -5 D lens in front of a randomly-selected
treated eye. The control eye had unrestricted vision through an open (no
lens) goggle frame. The goggle was removed for approximately three min
in dim illumination twice a day (at about 9:00 AM and at about 4:30 PM)
while the lens was cleaned. During lens cleaning, the animals were kept
in a darkened nest box to minimize exposure to visual stimuli. Badly
scratched lenses were replaced as needed while the animal was kept in
darkness (\lt 30 min).}

\subsection{Axial and refractive measures}

\p{At the time the pedestal was attached and at the end of the treatment
or recovery period, ocular component dimensions were measured under
anesthesia with A-scan ultrasound as described by Norton and McBrien
[5]. At the start and end of the treatment or recovery period,
non-cycloplegic measures of refractive state were taken on the animals
while they were awake using a Nidek ARK 700-A infrared autorefractor
[25]. This allowed us to assess the amount of myopia that developed
during the lens compensation period and, in the recovery group, the
amount of recovery that occurred in each animal. Prior studies have
found that non-cycloplegic awake autorefractor measures provide a valid
estimate of the amount of induced myopia in tree shrews. Actual values
for each eye differ from the cycloplegic measures by less than 1 D, and
the treated-eye versus control-eye differences are nearly identical
between non-cycloplegic and cycloplegic measures [6].}

\subsection{Two-dimensional gel electrophoresis}

\p{The animals were euthanized approximately 2 h into the light phase
with an overdose of sodium pentobarbital at the end of the treatment or
recovery period. Enucleated eyes were dissected in ice-cold 10 mM tris
and 250 mM sucrose (pH 7). Scleral tissue was immediately frozen in
liquid nitrogen, pulverized to a fine powder in a Teflon freezer mill
(B. Braun Biotech, Allentown, PA) while still frozen, and suspended in
500 \mu l extraction buffer (7 M urea, 2 M thiourea, 2% Pharmalyte 3-10,
4% CHAPS). Samples were then simultaneously reduced and alkylated at
room temperature for 90 min with 5 mM tributylphosphine (TBP) and 20 mM
4-vinyl pyridine (VP), respectively, before quenching the alkylation
with 20 mM DTT for 20 min. Cellular debris was pelleted at 21,000x g for
20 min at 4 \deg C. Supernatants (about 400 \mu l) were collected and
the extraction buffer was exchanged using Ultrafree 0.5 ml centrifugal
ultrafiltration devices (Millipore, Billerica, MA) with a 10 kDa cutoff
[26]. Samples were centrifuged at 12,000x g until a retentate volume of
about 50 \mu l was obtained, which was then diluted with an additional
500 \mu l extraction buffer. In total, the extraction buffer was
exchanged three times to ensure removal of any residual TBP or VP.
Protein yields were estimated using the 2D Quant Kit (GE Healthcare,
Piscataway, NJ).}

\p{For the first dimension, 100 \mu g scleral protein was diluted in
rehydration buffer (7 M urea, 2 M thiourea, 2% Pharmalyte 3-10) to give
a final CHAPS concentration of 0.5%. Samples were loaded onto 17 cm
immobilized pH gradient (IPG) pH 5-8 strips (Bio-Rad, Hercules, CA) by
active rehydration for 16 h at 20 \deg C using the PROTEAN IEF cell
(Bio-Rad, Hercules, CA). Electrode wicks were emplaced prior to
isoelectric focusing (IEF) for 4 h at 300 V followed by 3,500 V until a
total of 68,000 Vh had been reached. After IEF, IPG strips were
equilibrated (6 M urea, 50 mM tris pH 8.8, 30% glycerol, 2% SDS) for 30
min. The IPG strips were immediately affixed to the top of 12% SDS-PAGE
second dimension gels (20x25x0.1 cm). Assembled gels were
electrophoresed using the DALT\i{six} system (GE Healthcare, Piscataway,
NJ): 1 W per gel for 1 h followed by 13 W per gel for 6 h at 20 \deg C.}

\p{Following electrophoresis, the 2D gels were stained with Deep Purple
according to the manufacturer's instructions (GE Healthcare, Piscataway,
NJ) with minor modifications. Gels were rinsed extensively in H\sub{2}O
to prevent them from drying during scanning; gels were imaged at high
resolution (100 \mu m pixel size) using a Typhoon 8600 with 532 nm
excitation and the 610BP emission filter. The resulting protein profiles
from the treated and control eyes were compared for all animals in each
group at the UAB Proteomics \and\ Mass Spectrometry Shared Facility
using single stain analysis with intelligent noise correction algorithm
(INCA) processing by Progenesis 2D analysis software (Nonlinear
Dynamics, Newcastle upon Tyne, UK) to identify protein spots that were
shown to be differentially represented. Spots were considered to be
differentially expressed if there was a significant difference (p\lt
0.05; unpaired t-test) in normalized total spot volume between treated
and control eyes in the same direction in at least four of the five
animals in a group.}

\subsection{Mass spectrometry}

\p{For spot picking, gels were prepared as above except for loading 800
\mu g scleral protein per gel. Spots of interest were excised from the
gel and the plugs destained with three consecutive washes of 50 mM
ammonium bicarbonate/acetonitrile (1:1, v/v) for 30 min. Gel plugs were
then washed with 50 mM ammonium bicarbonate for 10 min prior to
digestion with freshly prepared trypsin (Trypsin Gold; Promega, Madison,
WI) at 37 \deg C overnight. Peptides were recovered from the gel plug
with two 100 \mu l washes in 5% formic acid/acetonitrile (1:1, v/v) for
30 min. The washes were combined and the solvent evaporated in a vacuum
centrifuge. Samples were resuspended in 10 \mu l 0.1% formic acid then
desalted and concentrated using ZipTips\sub{C18} (Millipore, Billerica,
MA) in preparation for MALDI-TOF (matrix-assisted laser
desorption/ionization time-of-flight) and LC-MSMS (liquid chromatography
tandem mass spectrometry) by the UAB Proteomics \and\ Mass Spectrometry
Shared Facility.}

\p{Desalted peptide samples were diluted 1/10 with a saturated solution
of \alpha-Cyano-4-hydroxycinnamic acid (CHCA) matrix and were applied to
an Applied Biosystems MALDI-TOF 96 well target plate and dried.
MALDI-TOF analyses were performed with a Voyager-DE PRO (Applied
Biosystems, Foster City, CA) in positive mode. Spectra were analyzed
using Voyager Data Explorer software; the resulting peptide mass
fingerprints were submitted via MASCOT (Matrix Science) to the Mass
Spectrometry protein sequence DataBase (MSDB) for protein
identification.}

\p{Tandem mass spectral analyses were performed with a Q-TOF2 mass
spectrometer (Micromass, Milford, MA) using electrospray ionization.
Liquid chromatography was performed using an Ultimate LC, Switchos
micro-column switching unit, and FAMOS autosampler (LC Packings,
Bannockburn, IL). The samples were concentrated on a 300 \mu m i.d.
C\sub{18} precolumn at a flow rate of 10 \mu l/min with 0.1% formic acid
and then flushed onto a 75 \mu m i.d. C\sub{18} column at 200 nl/min
with a gradient of 5-100% acetonitrile (0.1% formic acid) over 30 min. A
nano-LC interface was used to transfer the LC eluent into the Q-TOF
which was operated in automatic switching mode whereby multiply-charged
ions were subjected to MSMS if their intensities rose above six counts.
Tandem mass spectra were processed with MassLynx MaxEnt 3 software
(Micromass, Milford, MA) and submitted via ProteinLynx to the SwissProt
database for protein identification. All identities were manually
checked for accuracy.}

\results

\subsection{Changes during negative-lens compensation}

\p{At the end of the four day period of lens wear, all treated eyes had
partially compensated for the -5 D lens and were myopic relative to the
untreated fellow control eyes. \figref{1} shows the refractive values
for the control and treated eyes of each animal, corrected for the small
eye artifact [25,27] by subtracting 4 D. As a group, the treated eyes
were -3.5\pom 0.7 D (mean\pom SEM) myopic relative to the untreated
fellow control eyes. A-scan measures (not shown) confirmed the vitreous
chamber was elongated in the treated eyes relative to the control eyes.}

\p{Five protein spots were significantly differentially expressed
(downregulated) in the five pairs of eyes during myopia development.
\figref{2} shows the relative expression values of each differentially
expressed spot for treated and control eyes for each animal. All of the
differentially expressed spots were slightly downregulated in the
treated eye by 1.2 to 1.7 fold. Interestingly, the amount of
downregulation appeared to be related to the amount of myopia that
developed. The animal (0450) that developed the least myopia also had
the smallest differences in expression. However, there was not a
significant correlation. \figref{3} shows a representative
two-dimensional gel indicating the location of the five downregulated
protein spots.}

\p{All five of the protein spots were definitively identified by mass
spectrometry: spot 1 is pigment epithelium-derived factor (PEDF); spot 2
is procollagen I\alpha 1; spots 3 and 4 are thrombospondin I; spot 5 is
procollagen I\alpha 2. \tabref{1} shows the relative change in
expression level and peptide coverage for each of the proteins
identified. \figref{4} is an example of an LC-MSMS spectrograph for one
of the peptides from spot 1.}

\subsection{Differences during recovery}

\p{After 11-13 days of lens treatment, four of the five animals in the
recovery group had fully compensated for the -5 D lens. As shown in
\figref{5}, one animal (0570) developed less myopia. After four days of
recovery, varying amounts of refractive recovery had occurred, as in
previous studies [8,10,23].}

\p{Comparison of the recovering and control eye gels from the five
animals showed that three of the protein spots (the two procollagen I
spots and PEDF) that were downregulated during lens compensation were
not significantly different in the recovering versus control eyes. The
two thrombospondin I spots remained lower in abundance in the recovering
eyes (indicated by the \color{\blue}{blue} squares in \figref{3}).
Levels of a sixth spot (\color{\red}{red} square in \figref{3}) were
upregulated in the recovering eyes. \figref{6} shows the relative
protein levels in the recovering and control eyes for thrombospondin I
(spots 3 and 4) and for the upregulated spot 6. As illustrated in
\tabref{1}, this protein spot has been identified as 78 kDa
glucose-regulated protein (GRP 78; a member of the heat shock protein 70
family). Note that the animal (0570) that showed the least recovery did
not show an upregulation in spot 6. This was the only case when
differential expression was not seen in all five animals.}

\discussion

\p{The results have allowed us to make several conclusions about both
the proteomic methodology and its utility in the "neutral" approach to
detecting protein changes in tree shrew fibrous sclera. First, as
expected, the changes in protein levels were small in magnitude. This
was expected because a -5 D lens is a relatively mild stimulus, well
within the physiologically normal range of the eye, which merely shifts
the focal plane in the hyperopic direction. It is not a disruptive
stimulus that would be expected to cause on/off or massive upregulation
or downregulation of proteins and has previously been found to produce
similar (\lt two-fold) changes in mRNA levels [23].}

\p{Second, the results are consistent across animals. During lens
compensation, specific proteins were downregulated in all five animals,
and there was less downregulation in the animal that developed the least
myopia. A similar parallel was found during recovery. This raises
confidence in the precision of the method. Third, we have been able to
identify the tree shrew proteins whose levels changed. This initially
was a concern because there are currently few tree shrew entries in
sequence databases. However, the tree shrew proteins have sufficient
homology that we have been able to identify, unambiguously, all the
proteins that we have submitted to the UAB Mass Spectrometry and
Proteomics Core Facility. Fourth, we confirmed that we can find changes
in proteins that were found to change in previous studies such as type I
collagen, which studies have found to be less abundant during myopia
development in tree shrews [12,22]. Fifth, we have also found
downregulation of a protein, thrombospondin I, that was not previously
known to change in the sclera and that adds detail to our understanding
of the chain of signals that regulates scleral creep rate. A function of
thrombospondin I is to activate TGF-\beta\ [28]. The downregulation of
thrombospondin I is consistent with the reports of lowered levels of
TGF-\beta\ in tree shrew sclera [29] and the role of TGF-\beta\ which
stimulates, via the SMAD signal transduction pathway, the production of
type I collagen [30]. Thus, the lower thrombospondin I level may reduce
the levels of TGF-\beta\ which in turn may account for the lower levels
of type I procollagen that we found here and that have been reported
previously [14].}

\p{Finally, we have found differential changes in additional proteins,
PEDF and GRP 78 that, with thrombospondin I, affirm the importance of
using this proteomic approach. We would not have suspected that these
proteins would change in the sclera and would not have examined them if
we had restricted ourselves to using the "candidate protein" approach.
GRP 78 is a chaperone, facilitating multimeric protein assembly in the
endoplasmic reticulum that recognizes and binds to malfolded or
denatured proteins such as type I procollagen [31,32]. PEDF is known to
have important signaling characteristics. It has a high affinity to
various extracellular matrix components such as glycosaminoglycans and
collagen and is a possible modulator of the integrin-collagen
interaction [33,34]. Its expression is increased by
all-\i{trans-}retinoic acid [35] and is a substrate for MMP-2 and MMP-9
[36], and thus could be involved in the control of scleral
extensibility.}

\p{It is of interest that the number of proteins found to change is
perhaps smaller than might have been expected and that the proteins we
have identified differ from the ones identified with 2DGE in the chick
sclera [37]. For instance, it could be expected to see the previously
described MMP and TIMP (tissue inhibitor of metalloproteinase) changes
reproduced in this study. However, there are some limitations to the
methodology. For example, the active form of MMP-2 has an isoelectric
point (pI) of 5.0 and so cannot be resolved by the employed pH 5-8 IPG
gradient. The latent form of MMP-2 does have a slightly higher pI and so
should be present, but with a molecular weight of about 74 kDa, it would
be located just below spot 6 within the saturated region with its signal
inundated (\figref{3}). TIMP-3 would also not be displayed as both
active and latent forms of the protein have isoelectric points greater
than 9.0 and so neither can be resolved by this IPG gradient. A final
example is MT1-MMP which should have both active (MW about 54 kDa; pI
5.8) and latent (MW about 66 kDa; pI 7.6) forms displayed on the gel.
However, the 2DGE methodology cannot generally resolve membrane-bound
proteins such as MT1-MMP.}

\p{Furthermore, in this study, the proteins from the treated and control
eyes were in separate gels, which were aligned and compared using
software. The inherent variability across the gels may have limited the
ability to detect biological variation. Hence, the results of the
present study most likely provide a conservative survey of the number of
proteins whose levels change during negative-lens compensation and
recovery. Use of the Difference Gel Electrophoresis (DIGE) method in
which proteins from a treated eye, control eye, and a standard are each
labeled with a different spectrally resolvable, colocalizing fluorescent
dye and are all displayed on the same gel, which would reduce inter-gel
variability, may provide improved ability to detect biological
variation. Preliminary data using DIGE suggests that a larger group of
subtly differentially expressed proteins await discovery.}

\acknowledgements

\p{Supported by the EyeSight Foundation of Alabama and by National Eye
Institute Grants RO1 EY05922 and P30 EY03039 (CORE). The authors have no
commercial interest in the subject matter of the manuscript. We thank
Dr. John T. Siegwart, Jr. and Mr. Joel Robertson for assistance in the
preparation of the animals used in this study. Data in this paper were
presented at the Association for Research in Vision and Ophthalmology
(2006) E-abstract 1148 and at the 11th International Myopia Conference,
Ophthalmic and Physiological Optics (2006) 26 (suppl 1), 52.}

\references

\p{1. Cartmill M. Rethinking primate origins. Science 1974; 184:436-43.
\pubmed{4819676}}

\p{2. Luckett WP. Comparative biology and evolutionary relationships of
tree shrews. New York: Plenum Press; 1980.}

\p{3. Petry HM, Fox R, Casagrande VA. Spatial contrast sensitivity of
the tree shrew. Vision Res 1984; 24:1037-42. \pubmed{6506467}}

\p{4. McBrien NA, Norton TT. The development of experimental myopia and
ocular component dimensions in monocularly lid-sutured tree shrews
(Tupaia belangeri). Vision Res 1992; 32:843-52. Erratum in: Vision Res
1993 Nov; 33:2381. \pubmed{1604853}}

\p{5. Norton TT, McBrien NA. Normal development of refractive state and
ocular component dimensions in the tree shrew (Tupaia belangeri). Vision
Res 1992; 32:833-42. \pubmed{1604852}}

\p{6. Norton TT, Siegwart JT Jr, Amedo AO. Effectiveness of hyperopic
defocus, minimal defocus, or myopic defocus in competition with a
myopiagenic stimulus in tree shrew eyes. Invest Ophthalmol Vis Sci 2006;
47:4687-99. \pubmed{17065475}}

\p{7. Siegwart JT Jr, Norton TT. Goggles for controlling the visual
environment of small animals. Lab Anim Sci 1994; 44:292-4.
\pubmed{7933981}}

\p{8. Siegwart JT Jr, Norton TT. Regulation of the mechanical properties
of tree shrew sclera by the visual environment. Vision Res 1999;
39:387-407. \pubmed{10326144}}

\p{9. Norton TT, Siegwart JT Jr. Animal models of emmetropization:
matching axial length to the focal plane. J Am Optom Assoc 1995;
66:405-14. \pubmed{7560727}}

\p{10. Moring AG, Baker JR, Norton TT. Modulation of glycosaminoglycan
levels in tree shrew sclera during lens-induced myopia development and
recovery. Invest Ophthalmol Vis Sci 2007; 48:2947-56. \pubmed{17591859}}

\p{11. Amedo AO, Siegwart JT, Jr., Norton TT. The effect of age on
compensation to a minus lens and recovery in tree shrews. Invest
Ophthalmol Vis Sci 2007; 48:E-abstract 1029.}

\p{12. Norton TT, Rada JA. Reduced extracellular matrix in mammalian
sclera with induced myopia. Vision Res 1995; 35:1271-81.
\pubmed{7610587}}

\p{13. Gentle A, McBrien NA. Modulation of scleral DNA synthesis in
development of and recovery from induced axial myopia in the tree shrew.
Exp Eye Res 1999; 68:155-63. \pubmed{10068481}}

\p{14. McBrien NA, Lawlor P, Gentle A. Scleral remodeling during the
development of and recovery from axial myopia in the tree shrew. Invest
Ophthalmol Vis Sci 2000; 41:3713-9. \pubmed{11053267}}

\p{15. Gentle A, McBrien NA. Retinoscleral control of scleral
remodelling in refractive development: a role for endogenous FGF-2?
Cytokine 2002; 18:344-8. \pubmed{12160524}}

\p{16. McBrien NA, Gentle A. Role of the sclera in the development and
pathological complications of myopia. Prog Retin Eye Res 2003;
22:307-38. \pubmed{12852489}}

\p{17. Rada JA, Shelton S, Norton TT. The sclera and myopia. Exp Eye Res
2006; 82:185-200. \pubmed{16202407}}

\p{18. Norton TT, Metlapally R, Young TL. Myopia. In: Garner A,
Klintworth GK, editors. The pathobiology of ocular disease. New York:
Taylor \and\ Francis; In press 2007.}

\p{19. Guggenheim JA, McBrien NA. Form-deprivation myopia induces
activation of scleral matrix metalloproteinase-2 in tree shrew. Invest
Ophthalmol Vis Sci 1996; 37:1380-95. \pubmed{8641841}}

\p{20. Gentle A, Martin JE, McBrien NA. Differential expression of
collagen types I, III and V in the sclera of myopic eyes: A precursor to
fibril diameter changes? Invest Ophthalmol Vis Sci 2002; 43:ARVO
E-Abstract 2449.}

\p{21. Siegwart JT Jr, Norton TT. The time course of changes in mRNA
levels in tree shrew sclera during induced myopia and recovery. Invest
Ophthalmol Vis Sci 2002; 43:2067-75. \pubmed{12091398}}

\p{22. Gentle A, Liu Y, Martin JE, Conti GL, McBrien NA. Collagen gene
expression and the altered accumulation of scleral collagen during the
development of high myopia. J Biol Chem 2003; 278:16587-94.
\pubmed{12606541}}

\p{23. Siegwart JT Jr, Norton TT. Selective regulation of MMP and TIMP
mRNA levels in tree shrew sclera during minus lens compensation and
recovery. Invest Ophthalmol Vis Sci 2005; 46:3484-92. \pubmed{16186323}}

\p{24. Schippert R, Brand C, Schaeffel F, Feldkaemper MP. Changes in
scleral MMP-2, TIMP-2 and TGFbeta-2 mRNA expression after imposed myopic
and hyperopic defocus in chickens. Exp Eye Res 2006; 82:710-9.
\pubmed{16289164}}

\p{25. Norton TT, Wu WW, Siegwart JT Jr. Refractive state of tree shrew
eyes measured with cortical visual evoked potentials. Optom Vis Sci
2003; 80:623-31. \pubmed{14502042}}

\p{26. Smejkal GB, Li C, Robinson MH, Lazarev AV, Lawrence NP,
Chernokalskaya E. Simultaneous reduction and alkylation of protein
disulfides in a centrifugal ultrafiltration device prior to
two-dimensional gel electrophoresis. J Proteome Res 2006; 5:983-7.
\pubmed{16602706}}

\p{27. Glickstein M, Millodot M. Retinoscopy and eye size. Science 1970;
168:605-6. \pubmed{5436596}}

\p{28. Murphy-Ullrich JE, Poczatek M. Activation of latent TGF-beta by
thrombospondin-1: mechanisms and physiology. Cytokine Growth Factor Rev
2000; 11:59-69. \pubmed{10708953}}

\p{29. Jobling AI, Nguyen M, Gentle A, McBrien NA. Isoform-specific
changes in scleral transforming growth factor-beta expression and the
regulation of collagen synthesis during myopia progression. J Biol Chem
2004; 279:18121-6. \pubmed{14752095}}

\p{30. Overall CM, Wrana JL, Sodek J. Independent regulation of
collagenase, 72-kDa progelatinase, and metalloendoproteinase inhibitor
expression in human fibroblasts by transforming growth factor-beta. J
Biol Chem 1989; 264:1860-9. \pubmed{2536374}}

\p{31. Lee AS. Mammalian stress response: induction of the
glucose-regulated protein family. Curr Opin Cell Biol 1992; 4:267-73.
\pubmed{1599691}}

\p{32. Chessler SD, Byers PH. BiP binds type I procollagen pro alpha
chains with mutations in the carboxyl-terminal propeptide synthesized by
cells from patients with osteogenesis imperfecta. J Biol Chem 1993;
268:18226-33. \pubmed{8349698}}

\p{33. Becerra SP. Focus on Molecules: Pigment epithelium-derived factor
(PEDF). Exp Eye Res 2006; 82:739-40. \pubmed{16364294}}

\p{34. Ek ET, Dass CR, Choong PF. PEDF: a potential molecular
therapeutic target with multiple anti-cancer activities. Trends Mol Med
2006; 12:497-502. \pubmed{16962374}}

\p{35. Tombran-Tink J, Lara N, Apricio SE, Potluri P, Gee S, Ma JX,
Chader G, Barnstable CJ. Retinoic acid and dexamethasone regulate the
expression of PEDF in retinal and endothelial cells. Exp Eye Res 2004;
78:945-55. \pubmed{15051476}}

\p{36. Notari L, Miller A, Martinez A, Amaral J, Ju M, Robinson G, Smith
LE, Becerra SP. Pigment epithelium-derived factor is a substrate for
matrix metalloproteinase type 2 and type 9: implications for
downregulation in hypoxia. Invest Ophthalmol Vis Sci 2005; 46:2736-47.
\pubmed{16043845}}

\p{37. Bertrand E, Fritsch C, Diether S, Lambrou G, Muller D, Schaeffel
F, Schindler P, Schmid KL, van Oostrum J, Voshol H. Identification of
apolipoprotein A-I as a "STOP" signal for myopia. Mol Cell Proteomics
2006; 5:2158-66. \pubmed{16921168}}

\endreferences
}

\beginfigures

\figfile{1}{
\figtitle{1}{Refractive measures after negative-lens treatment}

\p{Refractive measures for the treated and control eyes after four days
of -5 D lens wear with the group mean (\pom SEM). At the end of the lens
wear period all treated eyes had partially compensated for the lens and
were myopic relative to the untreated fellow control eyes. Values are
corrected for the small-eye artifact by subtracting 4 D [25,27].}

\ctr{\gifimage{1}{700}{399}{22}}

}

\figfile{2}{
\figtitle{2}{Changes in protein expression during the development of lens-induced
myopia}

\p{Relative expression values of each differentially expressed spot for
treated and control eyes for each animal and the group mean (\pom SEM),
normalized to the mean control expression level. All of the
differentially expressed proteins were downregulated in the treated eye
compared to the control eye.}

\ctr{\gifimage{2}{600}{1593}{195}}

}

\figfile{3}{
\figtitle{3}{Representative scleral protein profile}

\p{An example silver stained two-dimensional gel within the pH 5-8 (pI)
and 15-250 kDa (MW) range to show the location of the protein spots
found to change. \color{\blue}{Blue} circles indicate the five proteins
that were significantly downregulated in the treated eye of all five
pairs of eyes. During recovery, two of these spots were still
downregulated (\color{\blue}{blue} squares) and an additional spot was
upregulated (\color{\red}{red} square).}

\ctr{\jpgimage{3}{700}{846}{247}}

}

\figfile{4}{
\figtitle{4}{Example of protein spot identification by mass spectrometry}

\p{Representative spectrum from LC-MSMS of the peptide identified as
SLSQQIENIR. The spectrum shows that data are of sufficient high quality
for definitive identification of the corresponding peptides.}

\ctr{\gifimage{4}{700}{494}{23}}

}

\figfile{5}{
\figtitle{5}{Refractive measures after treatment and recovery}

\p{Refractive values for the treated eyes after 11-13 days of -5 D lens
wear and after four days of recovery for each of the animals and the
group mean (\pom SEM). Values are corrected for the small-eye artifact
[25,27]. Control eyes (not shown) appeared unaffected by the treatment.}

\ctr{\gifimage{5}{700}{396}{40}}

}

\figfile{6}{
\figtitle{6}{Changes in protein expression during the recovery from lens-induced
myopia}

\p{Relative expression values of each differentially expressed spot for
recovering and control eyes for each animal and the group mean (\pom
SEM), normalized to the mean control expression level. Thrombospondin I
is downregulated while GRP78 is upregulated in the recovering eyes
compared to the control eyes.}

\ctr{\gifimage{6}{600}{963}{109}}

}

\begintables

\tabfile{1}{
\tabtitle{1}{Differentially expressed proteins identified from the scleral two-dimensional gels}

\p{Relative changes in expression as a percentage of the control
(mean\pom SEM), and p-values, for proteins determined to be
differentially expressed on the scleral 2D gels. Spots 1-5 were
down-regulated in the treated eye during myopia development. During
recovery from myopia spots 3 and 4 remained down-regulated while spots
1, 2, and 5 were no longer differentially expressed; spot 6 was
up-regulated in the treated eye. Differentially expressed proteins were
identified by mass spectrometry: peptides in black were determined by
LC-MSMS, those in \color{\red}{red} italics by MALDI-TOF. All peptides
matched human sequence entries in the Swiss-Prot database except for one
(*) which matched the bovine sequence (GenBank \genbankprot{Q95121}).}

\ctr{\gifimage{1}{800}{602}{56}}

}