Download Choroidal retinoic acid synthesis: a possible mediator between

Exp. Eye Res. (2000) 70, 519–527
doi : 10.1006!exer.1999.0813, available online at http :!!www.idealibrary.com on
Choroidal Retinoic Acid Synthesis : A Possible Mediator between
Refractive Error and Compensatory Eye Growth
J A M E S R. M E R T Z    J O S H W A L L M A N
Department of Biology, City College, City University of New York, 138th Street and Convent Avenue,
New York, NY 10031, U.S.A.
(Received Seattle 2 August 1999 and accepted in revised form 6 December 1999)
Research over the past two decades has shown that the growth of young eyes is guided by vision. If nearor far-sightedness is artificially imposed by spectacle lenses, eyes of primates and chicks compensate by
changing their rate of elongation, thereby growing back to the pre-lens optical condition. Little is known
about what chemical signals might mediate between visual effects on the retina and alterations of eye
growth. We present five findings that point to choroidal retinoic acid possibly being such a mediator.
First, the chick choroid can convert retinol into all-trans-retinoic acid at the rate of 11!3 pmoles mg
protein−! hr−!, compared to 1"3!0"3 for retina!RPE and no conversion for sclera. Second, those visual
conditions that cause increased rates of ocular elongation (diffusers or negative lens wear) produce a
sharp decrease in all-trans-retinoic acid synthesis to levels barely detectable with our assay. In contrast,
visual conditions which result in decreased rates of ocular elongation (recovery from diffusers or positive
lens wear) produce a four- to five-fold increase in the formation of all-trans-retinoic acid. Third, the
choroidal retinoic acid is found bound to a 28–32 kD protein. Fourth, a large fraction of the choroidal
retinoic acid synthesized in culture is found in a nucleus-enriched fraction of sclera. Finally, application
of retinoic acid to cultured sclera at physiological concentrations produced an inhibition of proteoglycan
production (as assessed by measuring sulfate incorporation) with a EC of 8#10−$ . These results show
"#
that the synthesis of choroidal retinoic acid is modulated by those visual manipulations that influence
ocular elongation and that this retinoic acid may reach the sclera in concentrations adequate to modulate
scleral proteoglycan formation.
! 2000 Academic Press
Key words : sclera ; choroid ; chick ; myopia ; hyperopia ; proteoglycan.
1. Introduction
The growth of the eye is regulated not only by the
developmental mechanisms common to all organs,
but also by feedback supplied by the visual input. This
regulation by visual feedback is demonstrated by the
rapid compensation that eyes of chicks and monkeys
show when hyperopia or myopia is imposed on them
by spectacle lenses (Irving, Sivak and Callender, 1992 ;
Schaeffel, Glasser and Howland, 1988 ; Smith and
Hung, 1999 ; Wildsoet and Wallman, 1995). It is
likely, at least in the case of chicks, that this
compensation is effected in part by modulation of the
growth of the sclera causing changes in the length
of the eye (Marzani and Wallman, 1997 ; Nickla,
Wildsoet and Wallman, 1997). Another influence of
the retina on eye growth is shown by the ocular
elongation and myopia provoked by form-deprivation
in all species studied (Troilo and Judge, 1993 ;
Wallman, Turkel and Trachtman, 1978 ; MarshTootle, 1989 ; Wiesel and Raviola, 1977). Although it
is unclear at present whether the same mechanisms
underlie this growth change as that provoked by
wearing spectacle lenses, both involve modulation of
* Address correspondence to : James R. Mertz, New England
College of Optometry, 424 Beacon St., Boston, MA 02115, U.S.A.
E-mail : mertzj"ne-optometry.edu
0014–4835!00!040519$09 $35.00!0
scleral growth (Rada and Matthews, 1994) and both
appear to be largely local to the region of retina
experiencing the altered vision (Diether and Schaeffel,
1997 ; Wallman et al., 1995).
Children tend to develop myopia during the school
years, with the incidence of myopia being associated
with the amount of reading or other near-work (Saw
et al., 1996 ; Zylbermann, Landau and Berson, 1993).
As reading imposes a slight hyperopic defocus on the
eye (accommodation does not entirely eliminate it)
(Rouse, Hutter and Shiftlett, 1984 ; Schaeffel, Weiss
and Seidel, 1999), interest has been drawn to the
possibility that the myopia attendant to heavy reading
is a normal compensatory response to this hyperopia
and thus is much like the spectacle lens compensation
shown by animals.
Much work has gone into identifying what chemical
signals might participate in the retina’s influence on
eye growth. Strong evidence points to acetylcholine, in
that antagonists to muscarinic acetylcholine receptors
reduce both form-deprivation myopia and the progression of clinical myopia in children (Kennedy,
1995 ; McBrien, Moghaddam and Reeder, 1993 ;
Stone, Lin and Laties, 1991), although it is quite
unclear which of the several ocular tissues on which
it acts is the relevant one (Fischer et al., 1998 ; Lind et
al., 1998). Changes in retinal dopamine are also
clearly correlated with form-deprivation myopia
! 2000 Academic Press
520
(Stone et al., 1989), which can be ameliorated in
chicks and monkeys by dopamine agonists (Iuvone et
al., 1991). Retinal dopamine may also be correlated
with the defocus caused by spectacle lenses (Guo et al.,
1995 ; Schaeffel et al., 1995), but the correlation is far
from perfect (Boelen et al., 1999 ; Schwahn and
Schaeffel, 1997). Finally, glucagonergic amacrine cells
show opposite changes in ZENK expression in chick
eyes wearing positive and negative spectacle lenses
(Fischer et al., 1999).
Whatever retinal chemical signals might be involved, a major puzzle is how the retinal signals could
cross the choroid, a tissue layer with high blood flow
and extensive fenestrated capillaries. We propose that
no retinal signal need cross the choroid. Instead the
retina may signal the choroid to produce retinoic acid,
which in turn influences the growth of the sclera.
Retinoic acid is an important signaling molecule in
the developing eye. In mice with knock-outs of the
retinoic acid receptors, the eyes are extremely small
with gross morphological defects in choroid and sclera
and retinal dysplasia (Grondona et al., 1996). In tissue
culture, retinoic acid is required for the survival and
differentiation of embryonic photoreceptor cells
(Kelley, Turner and Reh, 1994 ; Stenkamp, Gregory
and Adler, 1993). Furthermore, in both mice and
chicks, dorsal-ventral patterning of the retina is
determined by gradients of retinoic acid (McCaffery et
al., 1992 ; Mey, McCaffery and Drager, 1997). Finally,
in postnatal animals retinoic acid modulates the
expression of growth factors such as TGFβ (MacDonald
et al., 1995) and causes anatomical and physiological
light adaptation in carp retinas (Weiler et al., 1998).
In this paper we show a pattern of results which
argue that it is plausible that the choroid synthesizes
and releases retinoic acid at a level determined by the
visual circumstances of the retina ; this retinoic acid is
transported to the sclera and there inhibits extracellular matrix synthesis, thereby influencing ocular
elongation. In brief, our evidence is (a) the choroid
produces massive amounts of retinoic acid, the levels
of which are strongly affected by spectacle lenses or by
form-deprivation with diffusers ; (b) the choroidal
retinoic acid is released into medium where it is found
associated with specific proteins ; (c) scleral tissue
accumulates retinoic acid, but synthesizes little or
none ; (d) retinoic acid strongly inhibits the synthesis
of proteoglycans by the sclera in vitro at physiological
concentrations.
2. Materials and Methods
Visual Manipulations
To deprive eyes of form-vision, white translucent
vinyl diffusers were attached to the feathers around
one eye of 2-day-old chicks for 14 days. This results in
myopia and ocular elongation. Other chicks wore a
diffuser for 11 days, after which the diffuser was
J. R. M E R T Z A N D J. W A L L M A N
removed, and the eye was allowed to recover for 3
days. To impose myopia or hyperopia with spectacle
lenses, chicks 6–7 days old wore a $15 D or a %15
D lens, respectively, over one eye for 24 hr (details in
Wildsoet and Wallman, 1995).
Determination of in vitro Synthesis of Retinoic Acid
After pentobarbital killing, eyes were removed,
placed on a bed of ice, hemisected at the ora serrata,
and the vitreous removed. An 8 mm punch, taken from
the posterior pole of each eye, was placed in Dulbecco’s
Modified Eagle’s Medium (DMEM), and the retina and
most of the retinal pigment epithelium (RPE) were
removed. After any remaining RPE cells were brushed
off, the choroid was peeled from the sclera. The
isolated choroid, sclera, and retina plus RPE were each
kept in ice-cold DMEM prior to culture. Tissues were
incubated in 0"5 ml of the defined medium N2 of
Bottenstein and Sato (1979) containing 1 µCi ml−! alltrans-retinol ([20-methyl-%H] Dupont NEN Research
Products, Boston, MA, U.S.A.) for 2 hr at 37&C,
following which media and tissues were stored in
separate amber tubes at %80&C until analysis. Tissues
were homogenized in 0"5 ml PBS, an aliquot was
removed for protein determination by the Bradford
method (Biorad, Hercules, CA, U.S.A.), 0"6 ml of
isopropyl alcohol (containing 100 ng of unlabeled 9cis-, 13-cis- and all-trans-retinoic acid as carriers to
permit identification of the HPLC peaks of these
retinoids for isolation and counting) was added,
vortexed for 5 min, centrifuged at 10 000 g for 10
min and the supernatant mixed with 2 % acetic acid
solution to a final concentration of 25 % isopropyl
alcohol. The resulting solution was applied to a solid
phase extraction column (100 mg C8, Baxter
Scientific, Chicago, IL, U.S.A.), which had been
equilibrated in 1 % acetic acid solution, washed with 2
ml of 1 % acetic acid in water : methanol 60 : 40, then
washed with 100 µl of methanol and air dried for 1
min. Retinoic acid was eluted from the column with
2#300 µl of ethanol : methanol 75 : 25, dried with a
stream of N gas and dissolved in 100 µl of 1 % acetic
&
acid in water : acetonitrile 60 : 40 for HPLC injection.
Isomers of retinoic acid were separated using a reverse
phase column (ODS2, 25 cm#3"1 mm, 5 µm, with an
additional C18 guard column ; Keystone Scientific,
Keystone, PA, U.S.A.) and isocratic elution at 65&C at
a flow rate of 0"6 ml min−!. The mobile phase was
acetonitrile : 1 % acetic acid in water 65 : 35. The
absorbance of the elutant was monitored at 350 and
325 nm utilizing a diode array detector (Waters
Instruments, Medford, MA, U.S.A.). Each retinoid peak
was collected manually and the amount of radioactive
retinoid was determined with a liquid scintillation
counter (Beckman Instruments, LS 1800, Columbia,
MD, U.S.A.). To estimate background radioactivity,
samples equivalent in volume to those of the retinoic
acid peaks were collected prior to and after the retinoic
CHOROIDAL RETINOIC ACID AND EYE GROWTH
acid peaks. For each retinoic acid peak, the background radioactivity was subtracted, and the net
counts were corrected for percent recovery as determined by the retinoid standard for that peak. The
resulting counts were converted to pmol of retinoic
acid based on the specific activity of retinol, which was
determined by collecting the retinol HPLC peak from
tissue samples and determining its mass by absorbance
at 325 nm and its total radioactivity. The identification
of the radioactive retinoic acid isomers was confirmed
by normal phase HPLC, using two columns in tandem
array (Spherical Silica 7"5 cm#4"6 mm, 5 µm, Waters
Instruments, Medford, MA, U.S.A. and a 3 µm Supelcosil column, 7"5 cm#4"6 mm, Sigma, St. Louis,
MO, U.S.A.), with an isocratic elution at a flow rate of
1"8 ml min−!. The mobile phase was acetic acid :
acetonitrile : hexane 0"1 : 0"4 : 99"5. All culture experiments, retinoic acid extractions and HPLC analyses
were performed under either dim red light or monochromatic light of 589 nm.
Measurement of the in vivo Retinoic Acid Levels in
Sclera
Scleral tissue from five eyes (7 mm punches) was
pooled and homogenized with a Polytron homogenizer
(Brinkmann, Westbury, NY, U.S.A.) in 2 ml of water.
Lipids were extracted by adding 4 ml of chloroform :
methanol 2 : 1, the sample was vortexed for 1 min,
and an additional 2 ml of chloroform was added. The
sample was centrifuged at 2000 g for 15 min and the
chloroform layer was removed and reduced to 1 ml by
evaporation with a gentle stream of N gas. The acidic
&
lipids were isolated from the chloroform by solid phase
extraction using an aminopropyl-bonded silica gel
column, and retinoic acid levels were determined by
normal phase HPLC using a Waters model 2487 Dual
Wavelength Absorbance Detector or Waters Diode
array detector model 996 (Kurlandsky et al., 1995).
SDS Gel Electrophoresis of Binding Proteins
To examine whether the retinoic acid released into
the medium was associated with specific proteins, we
made choroid-conditioned medium from ten choroids
from eyes recovering from form-deprivation myopia by
incubating them with tritiated all-trans-retinol for 2
hr in N2 medium lacking catalase or conalbumin
(removed so that the only proteins in the conditioned
medium were from the choroidal tissue). The resulting
medium was divided into four 2"5 ml aliquots and
incubated for 1 hr with either 13-cis- or all-transretinoic acid or a mixture of all-trans- and 9-cisretinoic acid, in all cases at a concentration of 100 ng
ml−!, or with 10 µl of carrier (ethanol). Each aliquot
was concentrated to 100 µl with an Amicon Centriprep-10 filter at 4&C, and the concentrated conditioned
medium was UV-irradiated to cross-link the retinoic
acid to carrier proteins. To determine which proteins
521
were covalently labeled with retinoic acid, the crosslinked proteins were fractionated by SDS gel electrophoresis (Bernstein et al., 1995), the gel was sliced
into 2 mm pieces, digested with 30 % hydrogen
peroxide (0"5 ml slice−!) overnight at 37&C and the
radioactivity in each slice measured by scintillation
counting.
Inhibition of Proteoglycan Synthesis
To assess proteoglycan synthesis, we measured the
incorporation of precursor into glycosaminoglycans
(GAGs). Scleral punches, 8 mm in diameter, from
week old chicks were incubated with various concentrations of retinoic acid for 22 hr, pulsed with Na %"SO
&
'
for 2 hr, digested with protease K, and the GAGs
precipitated and counted as previously described
(Marzani and Wallman, 1997). Scleral punches from
untreated fellow eyes were incubated under the same
conditions with vehicle (ethanol). Inhibition was
assessed by dividing the synthetic rate in the experimental eye by that in the untreated fellow eye of
each animal.
3. Results
The Choroid, as well as the Retina, Converts Retinol to
Retinoic Acid
By separately incubating the tissue layers of the
chick posterior eye wall with tritiated all-trans-retinol
and analysing the metabolic products using HPLC, we
found that under our culture conditions the choroid
produces 20-fold more all-trans-retinoic acid (per
milligram protein) than does the retina plus retinal
pigment epithelium, established sources of ocular
retinoic acid (Table I) (Edwards et al., 1992 ;
McCaffery, Mey and Drager, 1996 ; Mey et al., 1997).
These synthetic levels are higher than those reported
in other tissues (Blaner and Olson, 1994). For example,
we found that the choroid produces 40 times more
retinoic acid than does chicken liver (liver produces
approximately 750 femtomoles mg protein−! hr−! ;
cf. Table I). After incubating the choroid with labeled
retinol, we found that the medium contained levels of
T I
Conversion of retinol to all-trans-retinoic acid in chick
eye
Retina$RPE
Choroid
Sclera
Femtomoles
mg protein−! hr−!
Femtomoles
8 mm punch−! hr−!
1330!300
10 600!3000
ND
600!140
730!150
ND
RPE, retinal pigment epithelium ; ND, not detected (detection limit
of the assay was 200 femtomoles mg protein−! hr−! and 25 femtomoles punch−! hr−!). Data presented (mean!..) are from separate experiments (n ' 4) for two columns.
522
J. R. M E R T Z A N D J. W A L L M A N
femtomoles punch−! hr−! in retina ; 51!32 femtomoles punch−! hr−! in medium, n ' 3), suggesting
that the choroid, unlike the retina, releases most of the
newly synthesized retinoic acid. Under our culture
conditions, the rate of synthesis appears to decline
over 90 min even though the tissue contains an
excess of substrate, but the original synthetic rate can
be repeatedly restored by putting the choroid into fresh
medium. A similar decline in synthesis has been seen
in cultured retinal Mu! ller cells (which synthesize
retinoic acid at levels similar to those we measured in
retinal punches) (Edwards et al., 1992).
F. 1. Plot of the quantity of retinoic acid synthesized as
a function of time after choroids from normal chicks, 10–14
days of age, were put into tissue culture with tritiated
retinol. After a short time, more retinoic acid is found in the
medium than in the choroidal tissue. The levels reached
an asymptote after 90 min. Each data point is the mean!..
of four animals.
F. 2. Rate of all-trans-retinoic acid synthesis by choroids
from eyes under different visual conditions. Bars are the
mean (!..) amount of newly synthesized retinoic acid
(isolated by HPLC) found in the choroid plus the medium. (a)
Wearing a translucent diffuser over one eye, which results in
myopia and increased rates of ocular elongation, lowers
retinoic acid synthesis to barely detectable levels. Removing
the diffuser (for 3 days) after 11 days, which decreases the
rate of ocular elongation, causes retinoic acid synthesis to
rise to four times the control levels [n ' 10 for both
experimental eyes ; n ' 20 for the combined control (untreated fellow) eyes, which did not differ significantly
between the two groups]. (b) Negative lenses (%15 D, worn
for 1 day at 6–7 days of age), which increase the rate of
ocular elongation, reduce the retinoic acid synthesis in the
choroid (n ' 5). (c) Positive lenses ($15 D worn for 1 day
at 6–7 days of age), which decrease the rate of ocular
elongation, increase retinoic acid synthesis, as in eyes with
diffusers removed (n ' 4).
all-trans-retinoic acid as high or higher than the tissue
(1"2-fold higher, not significant by unpaired t-test, P (
0"05 ; Fig. 1), and that this ratio remained constant
over 3 hr. In chick retinas, similarly cultured, the
amount of retinoic acid in the medium was less than
10 % of the amount found in the tissue (582!18
Visual Manipulations Alter the in vitro Synthesis of
Retinoic Acid by the Choroid
The amount of retinoic acid synthesized in vitro in
choroid depends on the visual conditions of the eyes
from which the choroid was taken. Visual formdeprivation by diffusers, which increases ocular
elongation and causes myopia, sharply reduces synthesis of retinoic acid to barely detectable levels [Fig.
2(A)]. In contrast, removing the diffuser (after 11 days
of wear), which reveals the eye’s myopia and results in
slowed ocular elongation and rapid recovery from the
myopia, causes the synthesis of all-trans-retinoic acid
to increase to a level four–five times that of controls
[Fig. 2(A)].
The same bi-directional modulation of choroidal
retinoic acid synthesis occurs after spectacle lenses are
worn for a day. Negative lenses, which shift the image
plane further back in the eye and increase the rate of
ocular elongation as the animal compensates for this
shift, cause decreased synthesis of all-trans-retinoic
acid [Fig. 2(B)] ; positive lenses, which shift the image
forward in the myopic direction and decrease the rate
of ocular elongation, result in increased synthesis of
all-trans-retinoic acid [Fig. 2(C)], as does the myopic
defocus resulting from removal of a diffuser [Fig. 2(A)].
The time course of the synthetic changes differs :
removal of diffusers causes the rate to nearly double in
6 hr, a significant increase [unpaired t-test, (P ) 0"01 ;
Fig 3(a) ; putting on diffusers causes a slow decline to
about half in 12 hr [unpaired t-test, P )0.05 ; Fig.
3(b)]. In both conditions, slightly more retinoic acid
was found in the medium than in the tissue, as was
the case in Fig. 1. The two visual conditions that cause
increased retinoic acid secretion also cause dramatic
thickening of the choroid (Wallman et al., 1995), with
an approximately similar time course (unpublished
data).
Thus choroidal all-trans-retinoic acid synthesis is
inversely related to the rate of ocular elongation,
rather than being associated with any particular
visual condition. It is high under conditions leading to
decreased ocular elongation (either positive lenses or
recovery from deprivation myopia), and low under
conditions leading to increased elongation (either
negative lenses or form-deprivation).
CHOROIDAL RETINOIC ACID AND EYE GROWTH
523
Choroidal Retinoic Acid in Medium is Found Associated
with Specific Proteins
The movement of retinoids through the body and
cells is facilitated by specific retinoid binding proteins
(Sporn, Roberts and Goodman, 1994). To determine if
the retinoic acid released by the choroid is associated
with a specific protein in culture medium, we used UV
photolysis (Bernstein et al., 1995), to link tritiated
retinoic acid released by choroidal tissue to associated
proteins. This linking resulted in the labeling of
proteins with a molecular weight range of approximately 28–32 kDa (Fig. 4). Incubation with an excess
of all-trans-retinoic acid prior to UV-cross-linking
results in a decrease in labeling ; incubation with a
F. 4. The tritiated retinoic acid secreted from the choroid
is bound to a protein or proteins with a molecular weight of
approximately 28 kDa, as indicated by the single peak of
radioactivity (") representing retinoic acid photolytically
cross-linked to binding proteins. The binding indicated by
this peak is not influenced by the presence, before crosslinking, of unlabeled 13-cis-retinoic acid (#). The binding is
reduced or abolished, however, by the presence, before crosslinking, of unlabeled 9-cis and all-trans-retinoic acid ( ),
both isomers observed in cultures of chick choroids. These
data are from one choroid, and are representative of three
separate experiments.
mixture of all-trans- and 9-cis-retinoic acid eliminated
the labeling. In contrast, pre-incubation with an
excess of 13-cis-retinoic acid did not displace the
labeling, nor did incubation with an isomeric mixture
of either retinol or retinaldehyde (data not shown).
These results indicate that the binding of retinoic acid
to these proteins is highly specific.
Determination of the in vivo Levels of Retinoic Acid in
Scleral Tissue
To assess the amount of retinoic acid found in the
sclera, we did HPLC on pooled samples from five eyes.
To confirm that the peak measured [Fig. 5(B)] is alltrans-retinoic acid we (a) showed that it eluted at the
same time as known all-trans-retinoic acid from Sigma
Chemical Co. (St. Louis, MO, U.S.A.) and (b) compared
the UV absorption spectrum of this peak to that of
known all-trans-retinoic acid and found that they were
identical (data not shown). The concentration of
retinoic acid in normal chick sclera is approximately
5"5#10−(  (n ' 3). This value was determined by
calculating the amount of retinoic acid from the area
under the retinoic acid peak in the HPLC profile and
calculating the volume of the punch of scleral tissue by
assuming an average thickness of 100 µm.
Retinoic Acid Alters Scleral Proteoglycan Formation
F. 3. Time course of (a) increase in choroidal all-transretinoic acid synthesis in eyes recovering from diffuser wear
and of (b) decline in synthesis in eyes with diffusers.
Recovering eyes showed a significant increase (compared to
control eyes) in synthetic rate after only 6 hr, but diffuserwear did not produce a significant decrease until 12 hr. Each
datapoint is the mean of four animals, except for the control
group (!), which contains all 40 control animals (there was
no significant difference among control groups), * P * 0"05 ;
** P * 0"01 (paired t-test relative to untreated fellow eyes).
As the size of the eye is determined by the sclera,
and scleral proteoglycan synthesis has been shown to
be correlated with changes in the rate of ocular
elongation (Rada, Thoft and Hassell, 1991), we
measured the effect of all-trans-retinoic acid on the
synthesis of scleral proteoglycans in vitro. This
treatment caused a dramatic dose-dependent decrease
in the incorporation of sulfate precursors into proteo-
524
F. 5. Endogenous retinoic acid in chick sclera. (a) HPLC
profiles of retinoic acid standards : peak 1 is 13-cis-retinoic
acid ; peak 2 is all-trans-retinoic acid. (b) HPLC profile of
acidic lipid fraction from five scleras, showing the presence of
all-trans-retinoic acid by a peak at the same time as the
standard in (a).
F. 6. Inhibition by retinoic acid of sulfate incorporation
into scleral glycosaminoglycans, a measure of proteoglycan
formation. Each data point (n + 5) is the mean percentage
[!S.E.(M.)] incorporation in a treated scleral punch of
that in an untreated identical punch from the fellow eye.
glycans (Fig. 6). The EC for all-trans-retinoic acid is
"#
8#10−( . At the endogenous levels of retinoic acid in
untreated scleras from normal eyes (5"5#10−( ),
J. R. M E R T Z A N D J. W A L L M A N
approximately 40 % of scleral proteoglycan synthesis
would be inhibited. If we assume that the rate of
choroidal retinoic acid released in vivo is similar to the
in vitro rate of 1"6–2"4 pmole cm−& hr−! (Table I), that
the normal choroid is 250 µm thick (Nickla, Wildsoet
and Wallman, 1998), that the sclera is 100 µm thick,
that retinoic acid diffuses freely in the choroid and
sclera and that very little of the retinoic acid released
into the choroid is removed by the blood, then the EC
"#
of retinoic acid would be attained within 5 min of
synthetic activity.
As choroidal cells release retinoic acid into culture
medium and retinoic acid alters scleral proteoglycan
formation, we measured the transfer to the sclera of
retinoic acid released into the medium by the choroid
in co-culture. Culturing choroid and sclera together
with tritiated retinol for 2 hr and then isolating a
nucleus-enriched fraction (Feeney-Burns and Berman,
1982) of the sclera, we found that this fraction was
highly enriched in all-trans-retinoic acid. Although
the scleral punch represents less than 1 % of the total
volume in culture (scleral volume was 3"8 µl of a total
volume of 500 µl), it accumulated more than 50 % of
the all-trans-retinoic acid released by the choroidal
tissue into the medium (290!50 femtomoles ; Table I
and Fig. 1).
4. Discussion
We have presented a set of results which link visual
inputs to changes in choroidal synthesis of retinoic
acid, and also link choroidal retinoic acid to the
modulation of scleral extracellular matrix proteoglycans. With respect to the first of these links, we found
that the choroid synthesizes large amounts of retinoic
acid, even more than the retina. Over half of the
retinoic acid synthesized by the choroid (but only 10 %
of that synthesized by the retina) is found in the
medium, suggesting either that it is actively secreted,
or that it passively diffuses out of the cells that
synthesize it, but is excluded from some compartments
of the choroid. In either case, some characteristic
particular to the choroid favors the release of retinoic
acid into the medium. The changes in choroidal
retinoic acid synthesis measured in vitro after lenswear appear to reflect changes that occur in vivo
because we have found comparable changes in the
levels of endogenous retinoic acid in the choroid when
birds wear positive or negative lenses for three days
(Mertz et al., 1999).
The modulation of choroidal retinoic acid by
specifically those visual conditions that affect ocular
elongation is compatible with retinoic acid being part
of a signal cascade from retina to sclera, modulating
ocular elongation and thereby influencing refractive
error. We find that two rather different visual
manipulations, wearing a diffuser and wearing a
negative lens, both cause increased ocular elongation
and both cause decreased synthesis of choroidal
CHOROIDAL RETINOIC ACID AND EYE GROWTH
retinoic acid. Furthermore, two other rather different
manipulations, wearing positive lenses and the removal of a previously worn diffuser, both cause
decreased ocular elongation and both cause increased
synthesis of choroidal retinoic acid.
To clarify whether these associations signify that
retinoic acid is part of a signal cascade leading to
altered eye growth or are epiphenomena of changes in
the level of neuronal activity in the retina, we
measured retinoic acid synthesis in two visual conditions which have large effects on ocular elongation,
but would be expected to have small effects on the
average level of retinal activity. First, we found that
brief daily periods of stroboscopic light at dawn and
dusk, which are known to inhibit the ocular elongation resulting from wearing diffusers (Nickla, 1996),
also prevent the decline in choroidal retinoic acid
synthesis usually found in eyes wearing diffusers
unpub. data. Second, we found that very brief episodes
of positive or negative lens-wear (2 min hr−! of lenswear for 3 days, darkness the rest of the time) produce
significant compensatory changes in the rate of ocular
elongation and also produce substantial and significant changes in the abundance of retinoic acid in the
choroid plus sclera. [Positive lenses decreased the rate
of ocular elongation and increased the abundance of
retinoic acid ; negative lenses did the opposite (Winawer, Mertz and Wallman, unpublished data)]. These
results further support the tight association between
the visual conditions that affect choroidal retinoic acid
and those that affect ocular elongation, suggesting
that the association may be causal, rather than
fortuitous.
In the retina, as well, visual conditions influence the
levels of retinoic acid. Form-deprivation causes a 21 %
increase in the retinoic acid content (Seko, Shimizu
and Tokoro, 1998). This change is much less than the
change in the level of synthesis in the choroid and is
in the opposite direction. We have preliminary
evidence that the wearing of positive or negative
spectacle lenses also modulates retinoic acid levels in
the retina, again in the opposite direction from the
changes in choroidal retinoic acid levels (Mertz et al.,
1999). Thus, retinal retinoic acid might also be a
participant in the same signaling cascade as the
choroidal retinoic acid. If this is so, it is unlikely that
retinal retinoic acid directly inhibits choroidal retinoic
acid synthesis because in our tissue culture system
exogenous retinoic acid does not inhibit choroidal
retinoic acid synthesis.
We propose that the choroid synthesizes retinoic
acid, which is transported to the sclera and modulates
its growth. This role for the choroid is not an obvious
one, first, because blood vessels predominate in the
choroid, and blood vessels have not been shown to be
a source of retinoic acid elsewhere in the body, and
second, because the abundant blood flow in the
choroid and the fenestrated capillaries of the choriocapillaris might be expected to carry away any small
525
molecules produced there. We suspect that the retinoic
acid is produced not by the blood vessels but by the
lamina fusca at the posterior margin of the choroid,
which places the source closer to the sclera than to the
choriocapillaris at the anterior side of the choroid.
Immunohistochemistry shows that the choroid contains two proteins associated with retinoid metabolism : cellular retinaldehyde binding protein, concentrated in the posterior choroid, and retinaldehyde
dehydrogenase, located throughout the choroid with
strong staining in the outer choroid (Fischer et al.,
2000). Furthermore, choroidal retinoic acid being
bound to a protein might reduce the diffusional losses.
Our results suggest that the transport of retinoic
acid to the sclera is facilitated by scleral tissue having
a special affinity for retinoic acid. When a piece of
sclera amounting to one percent of the volume of the
culture was cultured together with a piece of choroid,
the nucleus-enriched fraction of the sclera acquired in
2 hr about half of the retinoic acid synthesized.
Although it is difficult to extrapolate from these in
vitro results to estimate the concentrating ability of
the sclera in vivo, it seems plausible that if retinoic
acid is produced in the outer choroid, much of it
would end up in the nuclei of scleral cells.
As extracellular retinoic acid is always found
associated with retinoid binding proteins, the fact that
the retinoic acid synthesized by the choroid is bound to
a protein is also compatible with its transport to the
sclera. Although we know little about this protein, it
may be a novel retinoid-binding protein. Of the
previously described extracellular retinoic acid binding
proteins, albumin and interphotoreceptor retinoid
binding protein have higher molecular weight and
bind all isomers of retinoic acid, and lipocalin-type
prostaglandin D synthase, which binds retinoic acid
and is released from RPE cells into the interphotoreceptor space, is slightly smaller (21–26 vs 28–32
kDa) and binds retinaldehyde and 13-cis-retinoic acid
with a similar kD as that for retinoic acid (Tanaka et
al., 1997). The choroidal retinoic acid binding protein
may, however, be similar or identical to the 28 kD
protein found in the RPE by Bernstein et al. (1995).
Finally, we have shown that retinoic acid in vitro
strongly inhibits the synthesis of GAGs, which are
major constituents of the proteoglycans in the extracellular matrix. Others have shown that retinoic
acid in tissue culture inhibits proliferation of scleral
fibroblasts and chondrocytes, the two major cell types
in the avian sclera (Seko, Shimokawa and Tokoro,
1996). As the concentration of retinoic acid found in
the sclera of normal eyes is close to the center of the
dose-response curve (50 % inhibition), retinoic acid
might modulate proteoglycan synthesis bidirectionally. Thus, the levels of retinoic acid in eyes wearing
negative lenses might be low enough that the
decreased inhibition could account for part or all of the
increased proteoglycan synthesis observed with negative lenses (Marzani and Wallman, 1997 ; Nickla et al.,
526
1998) ; in addition, the increase in retinoic acid levels
with positive lenses could account for part or all of the
growth inhibition observed with these lenses. Therefore, sclera, which does not synthesize retinoic acid,
has the receptors for retinoic acid (both nuclear
retinoic acid receptors and retinoid#receptors)
(Fischer et al., 2000). These might be used to modulate
proteoglycan synthesis and cell division. Our results
imply that the source of this retinoic acid could be the
adjacent choroid.
If choroidal retinoic acid is a major intermediary
between altered visual conditions and subsequent
changes in ocular elongation, what retinal factors
might regulate its synthesis ? As mentioned above,
there are retinal signals, such as dopamine, acetylcholine, glucagon and retinoic acid, with links to
myopia. Perhaps a retinal signal such as one of these
induces the retinal pigment epithelium to secrete a
signal that modulates choroidal retinoic acid secretion,
which in turn modulates scleral growth and thereby
ocular elongation. If this is so, it is plausible that the
myopia that children commonly develop during the
school years may be associated with decreased
choroidal secretion of retinoic acid and that pharmacological stimulation of choroidal retinoic acid
synthesis might ameliorate myopic progression.
Acknowledgements
We are grateful to Daniel Marzani for his participation in
the proteoglycan experiments, to Cecilia Artacho for technical assistance and to Edward Gresick (CUNY Medical
School), Debora Nickla (N.E. College of Optometry), William
Blaner (Columbia University), Jonathan Winawer and Daniel
Marzani for reading earlier drafts of this manuscript. The
experiments presented here were prompted by unpublished
pilot experiments carried out with Debora Nickla (New
England College of Optometry, Boston, MA) and Ursula
Dra! ger (Harvard Medical School, Boston, MA). The work
reported here was supported by NIH grants EY2727 and
RR03060.
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