in Chick Eye - Semantic Scholar
Retinal Control on the Axial Length Mediated by Transforming Growth Factor-/? in Chick Eye Shigeru Honda, Shigeki Fujii, Yoshibumi Sekiya, and Misao Yamamoto
Purpose. To clarify retinal control on scleral growth in form-deprivation myopia (FDM) in the chick, the authors studied change in transforming growth factor-/? (TGF-/?) in the formdeprived eye and the effect of this growth factor on scleral cell proliferation and axial length. Methods. Change in TGF-/? in FDM in the chick was measured by reverse transcriptase polymerase chain reaction (RT-PCR), immunoblot, and immunohistochemistry. The effect of TGF-/? on ['1H]thymidine uptake of scleral chondrocytes was determined by organ culture. Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) were administered to determine the effect of TGF-/? activation on the axial length in normal and FDM eyes. Results. The content of TGF-/? messenger RNA (mRNA) and the active form of TGF-/? protein were reduced in FDM eyes compared with the control specimen. Reduced immunoreactivity of TGF-/? in FDM eyes was found in the photoreceptor layer. The TGF-/3 inhibited [sH]thymidine uptake into scleral chondrocytes. In the nondeprived eyes, the vitreous chamber depth and axial length were reduced after uPA treatment, whereas PAI-1 increased them. In the formdeprived eyes, uPA inhibited vitreous depth and axial length elongation, but PAI-1 had no effect. Conclusions. The authors' results suggest that TGF-/3 mediates retinal control of ocular growth. Axial elongation in FDM probably is correlated with the reduction of TGF-/? in the retina, retinal pigment epithelium, and choroid. The uPA and PAI-1 treatment controls the activation of TGF-/3 and affects axial length. Invest Ophthalmol Vis Sci. 1996;37:2519-2526.
i 1 orm-deprivation myopia (FDM) is characterized by axial elongation accompanied by a great myopic refractive error. 1 ' 2 In the chick, these changes result from remodeling of the sclera as shown by the progression of protein and DNA synthesis in scleral cells. 3 The retina has an important role in the control of axial length' 1 ; but, how it regulates scleral growth is not known. T h e dopaminergic system is one possible mechanism in retinal control 5 whose action is thought to involve a pathway mediated by the photoreceptor and retinal pigment epithelium (RPE). 6 Ehrlich et al 7 reported that the photoreceptor is the most important organ in the retina for the control of axial length.
Transforming growth factor-/? (TGF-/?) is one of the growth factors released from the photoreceptor and RPE in the retina.8'9 Under various conditions, TGF-/? has a broad range of functions,10"Ia although initially, it is synthesized and secreted as a latent form with no biologic activity.13 Seko et al14 suggested that expression of the protein that corresponds to the active form of TGF-/?2 in the retina-RPE-choroid increases in the FDM eye. In their study, the natural concentration of active TGF-/? in FDM eye could not be determined because the samples had been treated before measurement with an acidic solution that activates latent TGF-/? and affects the basal level of active TGF-/?. Therefore, the expression of active TGF-/? in FDM has yet to be determined.
From the DnjmHmr.nl of Ophthalmology, Kobe University School of Medicine, Kobe, Japan. Supported Iry a research grant for retinachoroi/tal idmphy from the Ministry and Welfare of Japan, and by Gmnl-in-Aid for Developmental Scientific Research (li)(2)07SS7110 from the Ministry of Education, Science and Culture of Japan. Submitted for publication March II, 1996; revised June 7, 1996; accepted July 12, 1996. Proprietary interest category: /V. Reprint requests: Shigem Honda, Department of Ophthalmology, Kobe University School of Medicine, Kusunolii-cho 7-5-2, Chuo-ku, Kobe 650, Japan.
We investigated the expression of messenger RNA (mRNA) and the active form of TGF-/? in the retinaRPE-choroid in FDM eyes and compared them with those in the control eyes. We also measured the level of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the retina-RPE-choroid in
Investigative Ophthalmology & Visual Science, November 1996, Vol. 37, No. 12 Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology & Visual Science, November 1996, Vol. 37, No. 12
FDM eyes because uPA and tPA are involved in the physiologic activation of TGF-/3 through elevation of the plasmin concentration 15 and PAI-1 inhibits this reaction.16 Next, we examined the action of TGF-/? on scleral cells in an organ culture study. Finally, to confirm the activation effect of TGF-/? in vivo, we administered plasminogen activator or plasminogen activator inhibitor to chick eyes and studied the effect on eye growth. MATERIALS AND METHODS Treatment of Chick Male white leghorn chicks were reared in a box in which the temperature was maintained at 30°C and under a 12-hour light-12-hour dark cycle. They were anesthetized by an intramuscular injection of ketamine hydrochloride (50 mg/kg) and xylazine (20 mg/ kg) before lid suture, drug administration, and eye measurement were carried out. An overdose of sodium pentobarbital (100 mg/kg) was given before excising the eye globes. All the chicks were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Drugs and Antibodies Porcine TGF-/3 (mixture of isoforms) and rabbit antiporcine TGF-/J polyclonal antibody, which can recognize TGF-/31, /31.2,/3% 03, and /?5 (described as TGF/3s), were obtained from R&D Systems (Minneapolis, MN). Recombinant basic fibroblast growth factor (bFGF) was from Oncogene Science (Uniondale, NY); uPA from Wakamoto (Osaka, Japan); and melanoma PAI-1 from American Diagnostica (Greenwich, CT). Rabbit antihuman uPA, antihuman tPA, and antihuman PAI-1 polyclonal antibody were purchased from Techno Clone GmbH (Vienna, Austria). Form Deprivation The right eyes of 2-day-old chicks underwent form deprivation by lid suture. The left eyes were used as the control specimens. After 10 days, the chicks were killed and both eyes enucleated. After enucleation, the axial length and equatorial diameter of each eye were measured, after which the eyes were subjected to RNA analysis, immunoblotting, and immunohistochemical studies. RNA Analysis The enucleated control and FDM eyes were divided into anterior and posterior halves, and the vitreous and sclera gently removed. Total RNA from the retinaRPE-choroid was isolated by guanidium isothiocyanate and cesium chloride centrifugation. Each 5 fig of transfer RNA (tRNA) from the control and FDM eyes
was subjected to random hexamer-primed first stranded complementary DNA (cDNA) synthesis with MMuLV reverse transcriptase (TOYOBO, Osaka, Japan). One of 10 samples of the cDNA product was subjected to 35 rounds of amplification with rTth DNA polymerase. We used primers designed from the mature chick TGF-/31 cDNA sequence17 (sense primer 5'GCCCTGGATACCAACTACTGC-3'; antisense primer 5'-GCTGCACTTGCAGGAACGCCAC-3'). The expected length of the PCR product was 336 base pair. The RT reaction and PCR cycle profile were RT, 5 minutes at 65°C, 60 minutes at 37°C, and 5 minutes at 75°C; PCR, 1 minute at 94°C, 1 minute at 60°C, and 1.5 minutes at 72°C. The 35 rounds of amplification were followed by final incubation for 10 minutes at 72°C, after which the products were separated by 1.5% agarose gel electrophoresis and bands of the expected length extracted. The resultant amplified PCR products were subcloned into the pGEM-T cloning vector (Promega Corporation, Madison, WI), then introduced into MgC12-competentXLl-Blue bacteria (Stratagene, La Jolla, CA). The amplified cDNA inserted was subjected to sequence analysis with Dye Primer Cycle Sequencing kits (Applied Biosystems, Norwalk, CT). Variation in the quantity of RNA subjected to RT-PCR was controlled by monitoring the expression of mRNA that corresponds to /3-actin, a housekeeping gene that is not affected by FDM treatment. Immunoblot Analysis Eye globes obtained from FDM chicks were divided into anterior and posterior halves. The retina-RPEchoroid was separated from the posterior eye cup and immediately homogenized on ice in buffer containing 0.1 M Tris/HCl (pH 7.4), 100 mM ethylenediaminetetraacetic acid, 500 fiM phenylmethylsulfonyl fluoride, and 20 fiM leupeptin. To avoid chemical activation of TGF-/?, no detergent was added until the addition of the sample buffer. Samples were centrifuged at 1000 X g for 20 minutes and the supernatants collected. Protein concentration was measured by the method of Lowry et al.18 The protein concentration of all samples was equalized with homogenizing buffer, after which sample buffer containing 5% sodium dodecyl sulfate and 10% 2-mercaptoethanol was added and the whole boiled. A 20-^ig portion of each protein sample was subjected to sodium dodecyl sulfate -polyacrylamide gel electrophoresis (a 15% gel for TGF-/? and 7.5% gels for PAs and PAI-1). The proteins that separated were transferred to a polyvinylidine difluoride filter (Millipore, Bedford, MA). Nonspecific binding sites on the polyvinylidine difluoride filter were blocked by incubation with 10% gelatin for 12 hours. Anti-TGF-/? (1:100), anti-uPA (1:100), anti-tPA (1:100), and anti-PAI-1 (1:100) antibodies, respectively, were allowed to react in phosphate-buf-
TGF-0 Mediates Retinal Control on the Axial Length TABLE 1.
Eye Measurements
Control FDM
Axial Length
Equatorial Dimension
8.73 ± 0.37 9.91 ± 1.10*
11.32 ± 0.37 11.54 ± 0.45
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antirabbit IgG antibody, after which they were observed with fluorescent light microscope. For the negative control specimens, the sections were incubated without the primary antibody and then incubated with the second antibody. Organ Culture
KDM = form-deprivation myopia. Values fire given as mm {mean ± SD [n = 14]). * P < 0.005.
fered saline (PBS) containing 0.03% Triton X-100, followed by goat antirabbit immunoglobulin G (IgG) antibody, and finally rabbit antiperoxidase antibody. The membrane was washed in PBS, after which enhanced chemiluminescence detection kit (Renaissance; DuPont NEN, Boston, MA) was used. Immunohistochemistry Enucleated eyes were fixed for 24 hours in 4% paraformaldehyde containing 0.2% picric acid. After fixation, the eyes were washed with 30% sucrose in 0.1 M PB, frozen then cut on a cryostat. Frozen sections 10 fj,m thick were prepared. Sections were treated first for 20 minutes in PBS containing 5% normal goat serum and 0.03% Triton X-100, then stained with and TGF-/3 antibody (1:1000) for 24 hours at 4°C in PBS containing 3% normal goat serum and 0.03% Triton X-100. The sections then were washed with PBS and incubated for 2 hours at 4°C in '/50 FITC-labeled goat
1
2
To show the effect of TGF-/? on the proliferation of scleral cells, an organ culture was made. Chick eyes were excised on days 3, 6,15, 21, and 28 after hatching for the time course study and on day 14 for the dose response study. To avoid degeneration of individual eye compartment, the enucleated eyes were washed with PBS, and the cornea and lens were removed and immediately incubated in Dulbecco's modified Eagle's medium (Gibco-BRL, Grand Island, NY) without serum or containing TGF-/3 (0.2, 2.0, 20.0 ng/ml). The enucleated eyes also were incubated with bFGF (2.0 and 20.0 ng/ml) to compare the effect of another growth factor. Organ culture was carried out at 37°C
1
kDa
-25.2
3
B
-56.6
-336bp
-61.5
B
-240bp
i. Polymerase chain reaction (PCR) products of transforming growth factor-/?l (TGF-/31) (A) and /?-actin (B) in Lhe retina-retinal pigment epithelium-choroid complex of a control (lane 2) and a form-deprived (lane 3) eye. The molecular weight marker is shown in lane 1. The molecular weight of each PCR product based on the molecular weight marker is indicated to the right. FIGURE
-40.4 2. Immunoblot analysis of transforming growth factor-/? (A), urokinase plasminogen activator (B), tissue plasminogen activator (C) and plasminogen activator inhibitor1 (D) in the retina-retinal pigment epithelium-choroid complex of a control (lane 1) and form-deprived (lane 2) eye. The molecular weight of each protein based on the molecular weight standard is indicated to the right. FIGURE
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3. Immunoreactivity of transforming growth factor-/? (TGF-/3) in the retina. (A) Control eye. (B) Form-deprived eye. Immunoreactivity of TGF-/?s is present throughout the photoreceptor layer. Intense immunoreactivity is found in the outer part of photoreceptor layer and inner part of retinal pigment epithelium (RPE) layer. Immunoreactivity in the photoreceptor layer in the form-deprived eye is decreased compared with the control eye. (C) Negative control section incubated without primary antibody shows faint nonspecific fluorescence in the outer part of photoreceptor layer and inner part of RPE layer. Bar = 10 /iin. SCL = sclera; CHO = choroid; RPE = retinal pigment epithelium layer; PRL = photoreceptor layer; ONL = outer nuclear layer.
FIGURE
for 12 hours in the presence of 95% air/5% carbon dioxide. The eyes were labeled with additional 10 /^Ci/ ml of [3H] thymidine (Amersham, Arlington Heights, IL) for 4 hours to measure cellular proliferative activity. The reaction was terminated in fixing solution that contained 4% paraformaldehyde and 0.2% picric acid. After dehydration in a graded series of ethanols and chloroform, 3 fim paraffin sections were prepared. The sections were deparaffinized, then dipped in Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY) diluted with a 1:1 volume of water, after which they were dried and placed in a light-tight box and left at 4°C for 2 weeks. At the end of this exposure, the sections were developed, fixed, and counterstained with hematoxylin-eosin. A section including posterior pole and optic nerve of each eye was chosen, and the number of cells in the cartilageous sclera was measured (4000 to 5000 cells were counted in each eye). Pharmacologic Experiment In Vivo A daily retrobulbar injection of PA (500 U/50 //I), PAI-1 (50 U/50 fA), or saline (50 fi\) was given behind the right eye globe of the lid-sutured and nonsutured chicks. As the control, the same volume of saline was injected behind the left eye globe of all of the chicks. After 10 days, the anterior chamber depth, lens thickness, vitreous chamber depth, and axial length of each eye were measured by A-mode ultrasonography.
Statistical Analysis The data obtained from the axial measurements of the eye, densitometric reading of Western blot study, as well as organ culture study and pharmacologic experiment were statistically analyzed using Student's ttest, and the significance were determined.
RESULTS Axial Length and Tissue Weight of FormDeprivation Myopia Eyes The axial length was increased in the FDM eyes in comparison to the control eyes, whereas the equatorial diameter showed no statistically significant change (Table 1). RNA Analysis Equal amounts of the 240 base pair /?-actin PCR products were detectable in the control and FDM eyes, but the intensity of the bands corresponding to the amplified TGF-/31 cDNA PCR products were markedly lower in FDM eyes than in control eyes (Fig. 1). The experiment was replicated three times, and the same results were obtained. Immunoblot Analysis The TGF-/3 antibody recognized a band at the molecular weight of 25.2 kDa as did the active TGF-/31 used
TGF-/3 Mediates Retinal Control on the Axial Length
2523 immunoreactivity was increased (Figs. 2B, 2C). The experiment was replicated six times. Densitometric analysis showed significant differences between the FDM eyes and control eyes in relation to active TGF/3s (63% of control eyes, P < 0.01), uPA (170% of control eyes, P < 0.01) and PAI-1 (66% of control eyes, P < 0.05) immunoreactivities, but not for the latent forms of TGF-/0S and tPA.
5-
Immunoreactivity of Transforming Growth Factor-/3 in the Retina
10
15
20
Time (days)
The immunoreactivity of TGF-/3s mainly was detected throughout the photoreceptor layer in the retina (Figs. 3A, 3B). A faint nonspecific fluorescence was found in the outer part of photoreceptor layer and inner part of RPE layer (Fig. 3C), probably due to the staining of the second antibodies in the intracellular or the extracellular matrix. The immunoreactivity in the outer part of RPE layer and in the choroid was not apparent. The immunoreactivity of TGF-/?s in the photoreceptor layer was decreased in the FDM eyes compared with the control eyes. Incorporation of [3H]thymidine [3H]thymidine was incorporated in the scleral chondrocytes. Its uptake was highest in the chick eyes at 21 days of age, and this uptake was inhibited significantly by TGF-/3 (Fig. 4A). The inhibition of [aH]thymidine incorporation by TGF-/3 was dose dependent (Fig. 4B). In contrast, bFGF stimulated the dosedependent incorporation of [sH]thymidine into chondrocytes (Fig. 4B). Change in Ocular Compartments After the Administration of uPA or PAI-1
FIGURE 4. Number of [3H]-labeled cells. (A) Time course of proliferation of scleral chondrocytes ( • ) and the effect of transforming growth factor-/? (TGF-/3) (20.0 ng/ml) (0). (B) Dose responses of TGF-/? and basic fibroblast growth factor on the proliferation of scleral chondrocytes. In all cases, values are mean ± standard error of the mean (n = 3). *P< 0.05; **P< 0.01. for the control eye (Fig. 2A). It also recognized bands at higher molecular weights that corresponded to the latent forms of TGF-/?s (data not shown). The uPA, tPA, and PAI-1 antibodies, respectively, recognized a band of molecular weight of 56.6, 61.5, and 40.4 kDa (Figs. 2B, 2C, 2D), the expected size of each protein. The immunoreaction of active TGF-/3s and PAI-1 was decreased in the FDM eyes compared with that of the control eyes (Figs. 2A, 2D), whereas uPA and tPA
The dimensions of the ocular compartments of the right eyes without form deprivation are listed in Table 2 and those with form deprivation are listed in Table 3. Comparing the control groups of form-deprived and nondeprived chick eyes, anterior chamber depth decreased in the form-deprived eyes compared with the nondeprived eyes, whereas lens thickness, vitreous chamber depth, and axial length increased (Tables 2 and 3). The changes in anterior chamber depth, vitreous chamber depth and axial length were significant (P < 0.01, < 0.001, < 0.05, respectively), but the change of lens thickness was not. Both in the form-deprived and nonform-deprived eyes, the anterior chamber depth and lens thickness of uPA-treated and PAI-1-treated eyes showed no significant changes compared with the control eyes (Tables 2 and 3). In the nonform-deprived eyes, the vitreous chamber depth and axial length of uPA-treated eyes decreased in comparison with the controls, whereas PAI1-treated eyes showed elongation (Table 2).
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TABLE 2. Dimensions of the Ocular Components of Nonform-Deprived Chick Eye Control uPA PAI-1
ACD
LT
VCD
AL
1.454 + 0.027 1.427 ± 0.046 1.432 ± 0.038
2.513 ± 0.092 2.539 ± 0.069 2.544 ± 0.036
5.123 ± 0.098 5.036 ± 0.038* 5.221 ± 0.103*
9.090 ± 0.091 9.001 ± 0.055* 9.197 ± 0.109*
ACD = anterior chamber depth; LT = lens thickness; VCD = vitreous chamber depth; AL = axial length; uPA = urokinase plasminogen activator; PAI-] = plasminogen activator inhibitor. Values arc given as mm (mean ± SD [n = 10]). *P < 0.05.
In the form-deprived eyes, the vitreous chamber depth and axial length were reduced significantly in uPA-treated eyes in comparison with the control eyes, but no marked change was found for the control eyes and PAI-1-treated eyes (Table 3). DISCUSSION The TGF-/3 is a growth factor that can inhibit, and in some cases stimulate, cell proliferation in a variety of cell lines.10"12 In die retina, TGF-/J is distributed in the outer segment of the photoreceptors and in the RPE.8'9 In our study, the immunoreactivity of TGF-/3s was seen predominantly throughout the photoreceptor layer. The intense immunoreactivity in the outer part of photoreceptor and inner part of RPE layer might represent the outer segments of photoreceptors or the microvilli of RPE. The decrease of immunoreactivity in the photoreceptor layer in FDM likely is because of the reduction of total concentration of TGF/3s including latent and active protein form because the antibody used in this study recognizes both molecules. This finding is supported by the data of RNA analysis and immunoblot study that showed both TGFPl mRNA and acdve protein forms of TGF-/?s in retin a-RPE-c ho ro id were reduced in FDM eyes against the control eyes. Because TGF-/31 and TGF-/32 stimulate the synthesis of their own mRNA in some cell lines including chick RPE,'9"21'23 decrease of active TGF-/31 and TGF-/32 likely results in the decrease of their synthesis. Like this, the decrease of active TGF-
/3s suggests the reduction of synthesis of TGF-/?s mRNA. At least, the synthesis of TGF-/31 mRNA was reduced. Presented data suggest that the turnover of TGF-/? is reduced in the retina-RPE-choroid in FDM eyes. A recent report using the enzyme-linked immunosorbent assay method has shown that the protein level of active TGF-/32 in retina-RPE-choroid increases in FDM eye.H The discrepancy between diis finding and our results may be because different TGF-/? isoforms are regulated differently under various conditions.21"23 Another possibility is the difference of method. Before measurement of the concentration of active TGF-/32, the samples were treated by acidic solution. Acidic solution strongly activated latent TGF(32 and might increase the concentration of active TGF-/02.2'1'25 Therefore, it is unlikely that their results represented the actual concentration of active TGF/32. In our immunoblot study, no detergent was added in the samples to avoid chemical activation of TGF/3s until the addition of sample buffer, which could represent the close concentration of active TGF-/?s naturally present in the sample of retina-RPE-choroid. Therefore, we concluded that the actual concentration of active TGF-/?s in the retina-RPE-choroid decreases in FDM eye. The tPA, uPA, and PAI-1 are known to be released from choroidal vascular endothelial cells2627 and play an important role in anticoagulation through the conversion of plasminogen to plasmin in the vessels. Recently, it has been shown that tPA is released from vascular endothelial cells into extravascular space in
TABLE 3. Dimensions
of the Ocular Components of Form-Deprived Chick Eye Control uPA PAI-1
ACD
LT
VCD
AL
1.204 ± 0.066 1.127 ± 0.079 1.177 ± 0.078
2.564 ± 0.024 2.579 ± 0.054 2.622 ± 0.045
5.535 ± 0.164 5.241 ± 0.199* 5.466 ± 0.397
9.303 ± 0.192 8.963 ± 0.178* 9.263 ± 0.387
ACD = anterior chamber depth; LT = lens thickness; VCD = vitreous chamber depth; AL = axial length; uPA = urokinase plasminogen activator; PAI-1 = plasminogen activator inhibitor. Values are given as mm (mean ± SD [n = 5]). * P < 0.05.
TGF-/3 Mediates Retinal Control on the Axial Length 28
the choroid. The RPE also is known to release uPA and PAI-1 into retinal and choroidal space.29"31 Released PAs are considered to be involved in the activation of TGF-/? in the choroid. Increase of uPA and decrease of PAI-1 in FDM eyes shown by immunoblot study can be explained with the reduction of active TGF-/?s because TGF-/? inhibits the synthesis of uPA and tPA while stimulating the synthesis of PAI-1 in some cell lines, including RPE.202732 Although increased uPA may participate in the activation of TGF/? in the retina-RPE-choroid in FDM,15 the effect is considered to be limited because the actual volume of active TGF-/?s in retina-RPE-choroid decreased in FDM eye. In addition, it is unlikely that increased uPA induces the axial elongation in FDM because additional uPA inhibited the axial elongation in form-deprived and nondeprived eyes. In this study, the potent axial elongation observed in form deprivation mainly was because of the increase of vitreous chamber depth as reported previously,12 whereas anterior chamber depth was reduced in formdeprived eyes. It may be explained with the compression of lid suture, but unfortunately details are unclear in the presented study. We consider that the effect of administrated uPA and PAI-1 on the vitreous chamber depth and axial length correlates with the activation of TGF-/?.15 Because the organ culture data presented here showed that TGF-/? inhibits DNA synthesis of scleral chondrocytes, the inhibition of uPA on the axial elongation is considered to be a result of the activation of TGF-/? in retina, RPE, choroid, and sclera. In nondeprived eyes, the opposite change was found by the administration of PAI-1, which inhibits the activation of TGF-/?.16 However, the effect of administrated PAI-1 in FDM eyes could not be detected, whereas uPA showed apparent inhibition on axial elongation in FDM. It may be explained that active TGF-/?s in retina-RPE-choroid is extremely low in FDM eye, so the inhibition of PAI1 on the activation of TGF-/? is not distinct, whereas the administration of uPA activates TGF-/? to get into the positive feedback regulation. Therefore, it is likely that uPA and PAI-1 affect the vitreous chamber depth and axial length through the activation or inactivation of TGF-/?. Rohrer et al33 have shown that the subconjunctival or intravitreous injection of TGF-/? shows no effect on the axial length. However, the administration of TGF-/? alters the natural distribution of this growth factor and may influence the proliferation of many kinds of cells that usually are not controlled by TGF-/?. In this study, the natural distribution of TGF/? remained unchanged because we injected only the activator of TGF-/?. Therefore, we assume that our results are different from those of Rohrer et al. However, there is another possibility that uPA activates some latent proteases311 that may participate directly
2525 in the remodeling of sclera, and PAI-1 inhibits the activation of them. Further investigation is necessary to disclose the effect of uPA and PAI-1 on the axial elongation. This study suggests that one of the retinal control on scleral growth seems to be mediated by TGF-/3 and its reduction results in the axial elongation in FDM of chick. Key Words axial length, form-deprivation myopia, plasminogen activator, plasminogen activator inhibitor, transforming growth factor-beta Acknowledgment The authors thank personnel at the Senjyu Co. Ltd. Creative Center for assisting with the treatment and measurement of animals. References 1. Wallman J, Turkel J, Trachtman J. Extreme myopia produced by modest changes in early visual experience. Science. 1978;201:1249-1251. 2. Pickett-Seltner RL, SivakJG, PasternakJJ. Experimentally induced myopia in chicks: morphometric and biochemical analysis during the first 14 days after hatching. Vision Res. 1988;28:323-328. 3. Christensen AM, Wallman J. Evidence that increased scleral growth underlies visual deprivation myopia in chicks. Invest Ophthalmol Vis Sci. 1991;32:2143-2150. 4. Wallman J. Retinal influences on sclera underlie visual deprivation myopia. In: Bock G, Widdows K, eds. Myopia and the Control of Eye Groiuth. Chiba Foundation Symposium 155. Chichester, UK: John Wiley and Sons; 1990:126-141. 5. Stone RA, Lin T, Lades AM, Iuvone M. Retinal dopamine and form-deprivation myopia. Proc Natl Acad Sci USA. 1989;86:704-706. 6. Rohrer B, Spira A, Stell WK. Apomorphine blocks form-deprivation myopia in chickens by a dopamine D2 receptor mechanism acting in retina or pigmented epithelium. Vis Neurosci. 1993; 10:447-453. 7. Ehrlich D, SattayasaiJ, ZappiaJ, Barrington M. Effects of selective neurotoxins on eye growth in the young chick. In: Bock G, Widdows K, eds. Myopia and the Control of Eye Growth. Chiba Foundation Symposium 155. Chichester, UK: John Wiley and Sons; 1990:6388. 8. Lutty G, Ikeda K, Chandler C, McLeod DS. Immunohistochemical localization of transforming growth factor-/? in human photoreceptors. Curr Eye Res. 1991; 10:61-74. 9. Tanihara H, Yoshida M, Matsumoto M, Yoshimura N. Identification of transforming growth factor-/? expressed in cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1993; 34:413-419. 10. Roberts AB, Anzano MA, Wakefield LM, et al. Type /? transforming growth factor: a bifunctional regulation of cellular growth. Proc Natl Acad Sci USA. 1985:82:119-123.
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11. Yang EY, Moses HL. Transforming growth factor /Siinduced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane. J Cell Biol. 1990; 111:731-741. 12. Hiraki Y, Inoue H, Hirai R, Kato Y, Suzuki F. Effect of transforming growth factor (3 on cell proliferation and glycosaminoglycan synthesis by rabbit growthplate chondrocytes in culture. Biochim Biophys Ada. 1988;969:91-99. 13. Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol. 1987; 105:1039-1045. 14. Seko Y, Shimokawa H, Tokoro T. Expression of bFGF and TGF-/32 in experimental myopia in chicks. Invest Ophthalmol Vis Sci. 1995;36:1183-1187. 15. Lyons RM, Gentry LE, Purchio AF, Moses HL. Mechanism of activation of latent recombinant transforming growth factor 01 by plasmin. / Cell Biol. 1990; 110: 1361-1367. 16. Wagner OF, Binder BR. Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor from the conditioned media of a human melanoma cell line./Biol Chem. 1986;261:14474-14481. 17. Jakowlew SB, Dillard PJ, Sporn MB, Roberts AB. Nucleotide sequence of chicken transforming growth factor-beta 1 (TGF-/J1). Nucleic Acids Res. 1988; 16:8730. 18. Lowry OH, Rosebrough NJ, Fair AL, Randall RJ. Protein measurement with the Folin-phenol reagent. / Biol Chem. 1951;193:265-275. 19. Van OSE, Roche NS, Flanders KC, Sporn MB, Roberts AB. Transforming growth factor beta 1 positively regulates its own expression in normal and transformed cells. J Biol Chem. 1988; 263:7741 -7746. 20. Fujii S, Honda S, Sekiya Y, Yamainoto M. Transforming growth factor 0 (TGF-/3) regulates mRNA expression of itself, plasminogen activator (PA) and plasminogen activator inhibitor (PAI) in cultured chick RPE cells. ARVO abstracts. Invest Ophthalmol Vis Sci. 1995; 36:477. 21. Bascom CC, Wolfshohl JR, Coffey RJ, et al. Complex regulation of transforming growth factor beta 1, beta 2, and beta 3 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors beta 1 and beta 2. Mol Cell Biol. 1989;9:5508-5515. 22. Jakowlew SB, CubertJ, Danielpour D, Sporn MB, Roberts AB. Differential regulation of the expression of transforming growth factor-beta mRNAs by growth factors and retinoic acid in chicken embryo chondro-
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33.
34.
cytes, myocytes, and fibroblasts. J Cell Physiol. 1992; 150:377-385. O'Reilly MA, Danielpour D, Roberts AB, Sporn MB. Regulation of expression of transforming growth factor-beta 2 by transforming growth factor-beta isoforms is dependent upon cell type. Growth Factors. 1992; 6:193-201. Lawrence DA, Pircher R, Jullien P. Conversion of a high molecular weight latent /3-TGF from chicken embryo fibroblasts into a low molecular weight active /3-TGF under acidic conditions. Biochem Biophys Res Commun. 1985;133:1026-1034. Brown PD, Wakefield LM, Levinson AD, Sporn MB. Physicochemical activation of recombinant latent transforming growth factor-beta's 1, 2, and 3. Growth Factors. 1990; 3:35-43. Lutty GA, Ikeda K, Chandler C, McLeod DS. Immunolocalization of tissue plasminogen activator in the diabetic and nondiabetic retina and choroid. Invest Ophthalmol Vis Sci. 1991;32:237-245. Hackett SF, Campochiaro PA. Modulation of plasminogen activator inhibitor-1 and urokinase in retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci. 1993; 34:2055-2061. WangY, Gillies C, Cone RE, O'Rourke J. Extravascular secretion of t-PA by the intact superfused choroid. Invest Ophthalmol Vis Sci. 1995; 36:1625-1632. Siren V, Stephens R, Salonen E-M, et al. Retinal pigment epithelial cells secrete urokinase-type plasminogen activator and its inhibitor PAI-1. Ophthalmic Res. 1992;24:203-212. Tripathi BJ, ParkJ, Tripathi RC. Extracellular release of tissue plasminogen activator is increased with the phagocytic activity of the retinal pigment epithelium. Invest Ophthalmol Vis Sci. 1989; 30:2470-2473. Siren V, Vaheri A, Immonen I. Secretion of plasminogen activators by cells cultured from subretinal fluid. Acta Ophthalmol. 1994; 72:218-222. Saksela O, Moscatelli D, Rifkin DB. The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells. / Cell Biol. 1987; 105:957-963. Rohrer B, Stell WK. Basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-/3) act as stop and go signals to modulate postnatal ocular growth in the chick. Exp Eye Res. 1994;58:553-562. Rada JA, Brenza HL. Increased latent gelatinase activity in the sclera of visually deprived chicks. Invest Ophthalmol Vis Sci. 1995; 36:1555-1565.
Purpose. To clarify retinal control on scleral growth in form-deprivation myopia (FDM) in the chick, the authors studied change in transforming growth factor-/? (TGF-/?) in the formdeprived eye and the effect of this growth factor on scleral cell proliferation and axial length. Methods. Change in TGF-/? in FDM in the chick was measured by reverse transcriptase polymerase chain reaction (RT-PCR), immunoblot, and immunohistochemistry. The effect of TGF-/? on ['1H]thymidine uptake of scleral chondrocytes was determined by organ culture. Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) were administered to determine the effect of TGF-/? activation on the axial length in normal and FDM eyes. Results. The content of TGF-/? messenger RNA (mRNA) and the active form of TGF-/? protein were reduced in FDM eyes compared with the control specimen. Reduced immunoreactivity of TGF-/? in FDM eyes was found in the photoreceptor layer. The TGF-/3 inhibited [sH]thymidine uptake into scleral chondrocytes. In the nondeprived eyes, the vitreous chamber depth and axial length were reduced after uPA treatment, whereas PAI-1 increased them. In the formdeprived eyes, uPA inhibited vitreous depth and axial length elongation, but PAI-1 had no effect. Conclusions. The authors' results suggest that TGF-/3 mediates retinal control of ocular growth. Axial elongation in FDM probably is correlated with the reduction of TGF-/? in the retina, retinal pigment epithelium, and choroid. The uPA and PAI-1 treatment controls the activation of TGF-/3 and affects axial length. Invest Ophthalmol Vis Sci. 1996;37:2519-2526.
i 1 orm-deprivation myopia (FDM) is characterized by axial elongation accompanied by a great myopic refractive error. 1 ' 2 In the chick, these changes result from remodeling of the sclera as shown by the progression of protein and DNA synthesis in scleral cells. 3 The retina has an important role in the control of axial length' 1 ; but, how it regulates scleral growth is not known. T h e dopaminergic system is one possible mechanism in retinal control 5 whose action is thought to involve a pathway mediated by the photoreceptor and retinal pigment epithelium (RPE). 6 Ehrlich et al 7 reported that the photoreceptor is the most important organ in the retina for the control of axial length.
Transforming growth factor-/? (TGF-/?) is one of the growth factors released from the photoreceptor and RPE in the retina.8'9 Under various conditions, TGF-/? has a broad range of functions,10"Ia although initially, it is synthesized and secreted as a latent form with no biologic activity.13 Seko et al14 suggested that expression of the protein that corresponds to the active form of TGF-/?2 in the retina-RPE-choroid increases in the FDM eye. In their study, the natural concentration of active TGF-/? in FDM eye could not be determined because the samples had been treated before measurement with an acidic solution that activates latent TGF-/? and affects the basal level of active TGF-/?. Therefore, the expression of active TGF-/? in FDM has yet to be determined.
From the DnjmHmr.nl of Ophthalmology, Kobe University School of Medicine, Kobe, Japan. Supported Iry a research grant for retinachoroi/tal idmphy from the Ministry and Welfare of Japan, and by Gmnl-in-Aid for Developmental Scientific Research (li)(2)07SS7110 from the Ministry of Education, Science and Culture of Japan. Submitted for publication March II, 1996; revised June 7, 1996; accepted July 12, 1996. Proprietary interest category: /V. Reprint requests: Shigem Honda, Department of Ophthalmology, Kobe University School of Medicine, Kusunolii-cho 7-5-2, Chuo-ku, Kobe 650, Japan.
We investigated the expression of messenger RNA (mRNA) and the active form of TGF-/? in the retinaRPE-choroid in FDM eyes and compared them with those in the control eyes. We also measured the level of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the retina-RPE-choroid in
Investigative Ophthalmology & Visual Science, November 1996, Vol. 37, No. 12 Copyright © Association for Research in Vision and Ophthalmology
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Investigative Ophthalmology & Visual Science, November 1996, Vol. 37, No. 12
FDM eyes because uPA and tPA are involved in the physiologic activation of TGF-/3 through elevation of the plasmin concentration 15 and PAI-1 inhibits this reaction.16 Next, we examined the action of TGF-/? on scleral cells in an organ culture study. Finally, to confirm the activation effect of TGF-/? in vivo, we administered plasminogen activator or plasminogen activator inhibitor to chick eyes and studied the effect on eye growth. MATERIALS AND METHODS Treatment of Chick Male white leghorn chicks were reared in a box in which the temperature was maintained at 30°C and under a 12-hour light-12-hour dark cycle. They were anesthetized by an intramuscular injection of ketamine hydrochloride (50 mg/kg) and xylazine (20 mg/ kg) before lid suture, drug administration, and eye measurement were carried out. An overdose of sodium pentobarbital (100 mg/kg) was given before excising the eye globes. All the chicks were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Drugs and Antibodies Porcine TGF-/3 (mixture of isoforms) and rabbit antiporcine TGF-/J polyclonal antibody, which can recognize TGF-/31, /31.2,/3% 03, and /?5 (described as TGF/3s), were obtained from R&D Systems (Minneapolis, MN). Recombinant basic fibroblast growth factor (bFGF) was from Oncogene Science (Uniondale, NY); uPA from Wakamoto (Osaka, Japan); and melanoma PAI-1 from American Diagnostica (Greenwich, CT). Rabbit antihuman uPA, antihuman tPA, and antihuman PAI-1 polyclonal antibody were purchased from Techno Clone GmbH (Vienna, Austria). Form Deprivation The right eyes of 2-day-old chicks underwent form deprivation by lid suture. The left eyes were used as the control specimens. After 10 days, the chicks were killed and both eyes enucleated. After enucleation, the axial length and equatorial diameter of each eye were measured, after which the eyes were subjected to RNA analysis, immunoblotting, and immunohistochemical studies. RNA Analysis The enucleated control and FDM eyes were divided into anterior and posterior halves, and the vitreous and sclera gently removed. Total RNA from the retinaRPE-choroid was isolated by guanidium isothiocyanate and cesium chloride centrifugation. Each 5 fig of transfer RNA (tRNA) from the control and FDM eyes
was subjected to random hexamer-primed first stranded complementary DNA (cDNA) synthesis with MMuLV reverse transcriptase (TOYOBO, Osaka, Japan). One of 10 samples of the cDNA product was subjected to 35 rounds of amplification with rTth DNA polymerase. We used primers designed from the mature chick TGF-/31 cDNA sequence17 (sense primer 5'GCCCTGGATACCAACTACTGC-3'; antisense primer 5'-GCTGCACTTGCAGGAACGCCAC-3'). The expected length of the PCR product was 336 base pair. The RT reaction and PCR cycle profile were RT, 5 minutes at 65°C, 60 minutes at 37°C, and 5 minutes at 75°C; PCR, 1 minute at 94°C, 1 minute at 60°C, and 1.5 minutes at 72°C. The 35 rounds of amplification were followed by final incubation for 10 minutes at 72°C, after which the products were separated by 1.5% agarose gel electrophoresis and bands of the expected length extracted. The resultant amplified PCR products were subcloned into the pGEM-T cloning vector (Promega Corporation, Madison, WI), then introduced into MgC12-competentXLl-Blue bacteria (Stratagene, La Jolla, CA). The amplified cDNA inserted was subjected to sequence analysis with Dye Primer Cycle Sequencing kits (Applied Biosystems, Norwalk, CT). Variation in the quantity of RNA subjected to RT-PCR was controlled by monitoring the expression of mRNA that corresponds to /3-actin, a housekeeping gene that is not affected by FDM treatment. Immunoblot Analysis Eye globes obtained from FDM chicks were divided into anterior and posterior halves. The retina-RPEchoroid was separated from the posterior eye cup and immediately homogenized on ice in buffer containing 0.1 M Tris/HCl (pH 7.4), 100 mM ethylenediaminetetraacetic acid, 500 fiM phenylmethylsulfonyl fluoride, and 20 fiM leupeptin. To avoid chemical activation of TGF-/?, no detergent was added until the addition of the sample buffer. Samples were centrifuged at 1000 X g for 20 minutes and the supernatants collected. Protein concentration was measured by the method of Lowry et al.18 The protein concentration of all samples was equalized with homogenizing buffer, after which sample buffer containing 5% sodium dodecyl sulfate and 10% 2-mercaptoethanol was added and the whole boiled. A 20-^ig portion of each protein sample was subjected to sodium dodecyl sulfate -polyacrylamide gel electrophoresis (a 15% gel for TGF-/? and 7.5% gels for PAs and PAI-1). The proteins that separated were transferred to a polyvinylidine difluoride filter (Millipore, Bedford, MA). Nonspecific binding sites on the polyvinylidine difluoride filter were blocked by incubation with 10% gelatin for 12 hours. Anti-TGF-/? (1:100), anti-uPA (1:100), anti-tPA (1:100), and anti-PAI-1 (1:100) antibodies, respectively, were allowed to react in phosphate-buf-
TGF-0 Mediates Retinal Control on the Axial Length TABLE 1.
Eye Measurements
Control FDM
Axial Length
Equatorial Dimension
8.73 ± 0.37 9.91 ± 1.10*
11.32 ± 0.37 11.54 ± 0.45
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antirabbit IgG antibody, after which they were observed with fluorescent light microscope. For the negative control specimens, the sections were incubated without the primary antibody and then incubated with the second antibody. Organ Culture
KDM = form-deprivation myopia. Values fire given as mm {mean ± SD [n = 14]). * P < 0.005.
fered saline (PBS) containing 0.03% Triton X-100, followed by goat antirabbit immunoglobulin G (IgG) antibody, and finally rabbit antiperoxidase antibody. The membrane was washed in PBS, after which enhanced chemiluminescence detection kit (Renaissance; DuPont NEN, Boston, MA) was used. Immunohistochemistry Enucleated eyes were fixed for 24 hours in 4% paraformaldehyde containing 0.2% picric acid. After fixation, the eyes were washed with 30% sucrose in 0.1 M PB, frozen then cut on a cryostat. Frozen sections 10 fj,m thick were prepared. Sections were treated first for 20 minutes in PBS containing 5% normal goat serum and 0.03% Triton X-100, then stained with and TGF-/3 antibody (1:1000) for 24 hours at 4°C in PBS containing 3% normal goat serum and 0.03% Triton X-100. The sections then were washed with PBS and incubated for 2 hours at 4°C in '/50 FITC-labeled goat
1
2
To show the effect of TGF-/? on the proliferation of scleral cells, an organ culture was made. Chick eyes were excised on days 3, 6,15, 21, and 28 after hatching for the time course study and on day 14 for the dose response study. To avoid degeneration of individual eye compartment, the enucleated eyes were washed with PBS, and the cornea and lens were removed and immediately incubated in Dulbecco's modified Eagle's medium (Gibco-BRL, Grand Island, NY) without serum or containing TGF-/3 (0.2, 2.0, 20.0 ng/ml). The enucleated eyes also were incubated with bFGF (2.0 and 20.0 ng/ml) to compare the effect of another growth factor. Organ culture was carried out at 37°C
1
kDa
-25.2
3
B
-56.6
-336bp
-61.5
B
-240bp
i. Polymerase chain reaction (PCR) products of transforming growth factor-/?l (TGF-/31) (A) and /?-actin (B) in Lhe retina-retinal pigment epithelium-choroid complex of a control (lane 2) and a form-deprived (lane 3) eye. The molecular weight marker is shown in lane 1. The molecular weight of each PCR product based on the molecular weight marker is indicated to the right. FIGURE
-40.4 2. Immunoblot analysis of transforming growth factor-/? (A), urokinase plasminogen activator (B), tissue plasminogen activator (C) and plasminogen activator inhibitor1 (D) in the retina-retinal pigment epithelium-choroid complex of a control (lane 1) and form-deprived (lane 2) eye. The molecular weight of each protein based on the molecular weight standard is indicated to the right. FIGURE
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3. Immunoreactivity of transforming growth factor-/? (TGF-/3) in the retina. (A) Control eye. (B) Form-deprived eye. Immunoreactivity of TGF-/?s is present throughout the photoreceptor layer. Intense immunoreactivity is found in the outer part of photoreceptor layer and inner part of retinal pigment epithelium (RPE) layer. Immunoreactivity in the photoreceptor layer in the form-deprived eye is decreased compared with the control eye. (C) Negative control section incubated without primary antibody shows faint nonspecific fluorescence in the outer part of photoreceptor layer and inner part of RPE layer. Bar = 10 /iin. SCL = sclera; CHO = choroid; RPE = retinal pigment epithelium layer; PRL = photoreceptor layer; ONL = outer nuclear layer.
FIGURE
for 12 hours in the presence of 95% air/5% carbon dioxide. The eyes were labeled with additional 10 /^Ci/ ml of [3H] thymidine (Amersham, Arlington Heights, IL) for 4 hours to measure cellular proliferative activity. The reaction was terminated in fixing solution that contained 4% paraformaldehyde and 0.2% picric acid. After dehydration in a graded series of ethanols and chloroform, 3 fim paraffin sections were prepared. The sections were deparaffinized, then dipped in Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY) diluted with a 1:1 volume of water, after which they were dried and placed in a light-tight box and left at 4°C for 2 weeks. At the end of this exposure, the sections were developed, fixed, and counterstained with hematoxylin-eosin. A section including posterior pole and optic nerve of each eye was chosen, and the number of cells in the cartilageous sclera was measured (4000 to 5000 cells were counted in each eye). Pharmacologic Experiment In Vivo A daily retrobulbar injection of PA (500 U/50 //I), PAI-1 (50 U/50 fA), or saline (50 fi\) was given behind the right eye globe of the lid-sutured and nonsutured chicks. As the control, the same volume of saline was injected behind the left eye globe of all of the chicks. After 10 days, the anterior chamber depth, lens thickness, vitreous chamber depth, and axial length of each eye were measured by A-mode ultrasonography.
Statistical Analysis The data obtained from the axial measurements of the eye, densitometric reading of Western blot study, as well as organ culture study and pharmacologic experiment were statistically analyzed using Student's ttest, and the significance were determined.
RESULTS Axial Length and Tissue Weight of FormDeprivation Myopia Eyes The axial length was increased in the FDM eyes in comparison to the control eyes, whereas the equatorial diameter showed no statistically significant change (Table 1). RNA Analysis Equal amounts of the 240 base pair /?-actin PCR products were detectable in the control and FDM eyes, but the intensity of the bands corresponding to the amplified TGF-/31 cDNA PCR products were markedly lower in FDM eyes than in control eyes (Fig. 1). The experiment was replicated three times, and the same results were obtained. Immunoblot Analysis The TGF-/3 antibody recognized a band at the molecular weight of 25.2 kDa as did the active TGF-/31 used
TGF-/3 Mediates Retinal Control on the Axial Length
2523 immunoreactivity was increased (Figs. 2B, 2C). The experiment was replicated six times. Densitometric analysis showed significant differences between the FDM eyes and control eyes in relation to active TGF/3s (63% of control eyes, P < 0.01), uPA (170% of control eyes, P < 0.01) and PAI-1 (66% of control eyes, P < 0.05) immunoreactivities, but not for the latent forms of TGF-/0S and tPA.
5-
Immunoreactivity of Transforming Growth Factor-/3 in the Retina
10
15
20
Time (days)
The immunoreactivity of TGF-/3s mainly was detected throughout the photoreceptor layer in the retina (Figs. 3A, 3B). A faint nonspecific fluorescence was found in the outer part of photoreceptor layer and inner part of RPE layer (Fig. 3C), probably due to the staining of the second antibodies in the intracellular or the extracellular matrix. The immunoreactivity in the outer part of RPE layer and in the choroid was not apparent. The immunoreactivity of TGF-/?s in the photoreceptor layer was decreased in the FDM eyes compared with the control eyes. Incorporation of [3H]thymidine [3H]thymidine was incorporated in the scleral chondrocytes. Its uptake was highest in the chick eyes at 21 days of age, and this uptake was inhibited significantly by TGF-/3 (Fig. 4A). The inhibition of [aH]thymidine incorporation by TGF-/3 was dose dependent (Fig. 4B). In contrast, bFGF stimulated the dosedependent incorporation of [sH]thymidine into chondrocytes (Fig. 4B). Change in Ocular Compartments After the Administration of uPA or PAI-1
FIGURE 4. Number of [3H]-labeled cells. (A) Time course of proliferation of scleral chondrocytes ( • ) and the effect of transforming growth factor-/? (TGF-/3) (20.0 ng/ml) (0). (B) Dose responses of TGF-/? and basic fibroblast growth factor on the proliferation of scleral chondrocytes. In all cases, values are mean ± standard error of the mean (n = 3). *P< 0.05; **P< 0.01. for the control eye (Fig. 2A). It also recognized bands at higher molecular weights that corresponded to the latent forms of TGF-/?s (data not shown). The uPA, tPA, and PAI-1 antibodies, respectively, recognized a band of molecular weight of 56.6, 61.5, and 40.4 kDa (Figs. 2B, 2C, 2D), the expected size of each protein. The immunoreaction of active TGF-/3s and PAI-1 was decreased in the FDM eyes compared with that of the control eyes (Figs. 2A, 2D), whereas uPA and tPA
The dimensions of the ocular compartments of the right eyes without form deprivation are listed in Table 2 and those with form deprivation are listed in Table 3. Comparing the control groups of form-deprived and nondeprived chick eyes, anterior chamber depth decreased in the form-deprived eyes compared with the nondeprived eyes, whereas lens thickness, vitreous chamber depth, and axial length increased (Tables 2 and 3). The changes in anterior chamber depth, vitreous chamber depth and axial length were significant (P < 0.01, < 0.001, < 0.05, respectively), but the change of lens thickness was not. Both in the form-deprived and nonform-deprived eyes, the anterior chamber depth and lens thickness of uPA-treated and PAI-1-treated eyes showed no significant changes compared with the control eyes (Tables 2 and 3). In the nonform-deprived eyes, the vitreous chamber depth and axial length of uPA-treated eyes decreased in comparison with the controls, whereas PAI1-treated eyes showed elongation (Table 2).
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TABLE 2. Dimensions of the Ocular Components of Nonform-Deprived Chick Eye Control uPA PAI-1
ACD
LT
VCD
AL
1.454 + 0.027 1.427 ± 0.046 1.432 ± 0.038
2.513 ± 0.092 2.539 ± 0.069 2.544 ± 0.036
5.123 ± 0.098 5.036 ± 0.038* 5.221 ± 0.103*
9.090 ± 0.091 9.001 ± 0.055* 9.197 ± 0.109*
ACD = anterior chamber depth; LT = lens thickness; VCD = vitreous chamber depth; AL = axial length; uPA = urokinase plasminogen activator; PAI-] = plasminogen activator inhibitor. Values arc given as mm (mean ± SD [n = 10]). *P < 0.05.
In the form-deprived eyes, the vitreous chamber depth and axial length were reduced significantly in uPA-treated eyes in comparison with the control eyes, but no marked change was found for the control eyes and PAI-1-treated eyes (Table 3). DISCUSSION The TGF-/3 is a growth factor that can inhibit, and in some cases stimulate, cell proliferation in a variety of cell lines.10"12 In die retina, TGF-/J is distributed in the outer segment of the photoreceptors and in the RPE.8'9 In our study, the immunoreactivity of TGF-/3s was seen predominantly throughout the photoreceptor layer. The intense immunoreactivity in the outer part of photoreceptor and inner part of RPE layer might represent the outer segments of photoreceptors or the microvilli of RPE. The decrease of immunoreactivity in the photoreceptor layer in FDM likely is because of the reduction of total concentration of TGF/3s including latent and active protein form because the antibody used in this study recognizes both molecules. This finding is supported by the data of RNA analysis and immunoblot study that showed both TGFPl mRNA and acdve protein forms of TGF-/?s in retin a-RPE-c ho ro id were reduced in FDM eyes against the control eyes. Because TGF-/31 and TGF-/32 stimulate the synthesis of their own mRNA in some cell lines including chick RPE,'9"21'23 decrease of active TGF-/31 and TGF-/32 likely results in the decrease of their synthesis. Like this, the decrease of active TGF-
/3s suggests the reduction of synthesis of TGF-/?s mRNA. At least, the synthesis of TGF-/31 mRNA was reduced. Presented data suggest that the turnover of TGF-/? is reduced in the retina-RPE-choroid in FDM eyes. A recent report using the enzyme-linked immunosorbent assay method has shown that the protein level of active TGF-/32 in retina-RPE-choroid increases in FDM eye.H The discrepancy between diis finding and our results may be because different TGF-/? isoforms are regulated differently under various conditions.21"23 Another possibility is the difference of method. Before measurement of the concentration of active TGF-/32, the samples were treated by acidic solution. Acidic solution strongly activated latent TGF(32 and might increase the concentration of active TGF-/02.2'1'25 Therefore, it is unlikely that their results represented the actual concentration of active TGF/32. In our immunoblot study, no detergent was added in the samples to avoid chemical activation of TGF/3s until the addition of sample buffer, which could represent the close concentration of active TGF-/?s naturally present in the sample of retina-RPE-choroid. Therefore, we concluded that the actual concentration of active TGF-/?s in the retina-RPE-choroid decreases in FDM eye. The tPA, uPA, and PAI-1 are known to be released from choroidal vascular endothelial cells2627 and play an important role in anticoagulation through the conversion of plasminogen to plasmin in the vessels. Recently, it has been shown that tPA is released from vascular endothelial cells into extravascular space in
TABLE 3. Dimensions
of the Ocular Components of Form-Deprived Chick Eye Control uPA PAI-1
ACD
LT
VCD
AL
1.204 ± 0.066 1.127 ± 0.079 1.177 ± 0.078
2.564 ± 0.024 2.579 ± 0.054 2.622 ± 0.045
5.535 ± 0.164 5.241 ± 0.199* 5.466 ± 0.397
9.303 ± 0.192 8.963 ± 0.178* 9.263 ± 0.387
ACD = anterior chamber depth; LT = lens thickness; VCD = vitreous chamber depth; AL = axial length; uPA = urokinase plasminogen activator; PAI-1 = plasminogen activator inhibitor. Values are given as mm (mean ± SD [n = 5]). * P < 0.05.
TGF-/3 Mediates Retinal Control on the Axial Length 28
the choroid. The RPE also is known to release uPA and PAI-1 into retinal and choroidal space.29"31 Released PAs are considered to be involved in the activation of TGF-/? in the choroid. Increase of uPA and decrease of PAI-1 in FDM eyes shown by immunoblot study can be explained with the reduction of active TGF-/?s because TGF-/? inhibits the synthesis of uPA and tPA while stimulating the synthesis of PAI-1 in some cell lines, including RPE.202732 Although increased uPA may participate in the activation of TGF/? in the retina-RPE-choroid in FDM,15 the effect is considered to be limited because the actual volume of active TGF-/?s in retina-RPE-choroid decreased in FDM eye. In addition, it is unlikely that increased uPA induces the axial elongation in FDM because additional uPA inhibited the axial elongation in form-deprived and nondeprived eyes. In this study, the potent axial elongation observed in form deprivation mainly was because of the increase of vitreous chamber depth as reported previously,12 whereas anterior chamber depth was reduced in formdeprived eyes. It may be explained with the compression of lid suture, but unfortunately details are unclear in the presented study. We consider that the effect of administrated uPA and PAI-1 on the vitreous chamber depth and axial length correlates with the activation of TGF-/?.15 Because the organ culture data presented here showed that TGF-/? inhibits DNA synthesis of scleral chondrocytes, the inhibition of uPA on the axial elongation is considered to be a result of the activation of TGF-/? in retina, RPE, choroid, and sclera. In nondeprived eyes, the opposite change was found by the administration of PAI-1, which inhibits the activation of TGF-/?.16 However, the effect of administrated PAI-1 in FDM eyes could not be detected, whereas uPA showed apparent inhibition on axial elongation in FDM. It may be explained that active TGF-/?s in retina-RPE-choroid is extremely low in FDM eye, so the inhibition of PAI1 on the activation of TGF-/? is not distinct, whereas the administration of uPA activates TGF-/? to get into the positive feedback regulation. Therefore, it is likely that uPA and PAI-1 affect the vitreous chamber depth and axial length through the activation or inactivation of TGF-/?. Rohrer et al33 have shown that the subconjunctival or intravitreous injection of TGF-/? shows no effect on the axial length. However, the administration of TGF-/? alters the natural distribution of this growth factor and may influence the proliferation of many kinds of cells that usually are not controlled by TGF-/?. In this study, the natural distribution of TGF/? remained unchanged because we injected only the activator of TGF-/?. Therefore, we assume that our results are different from those of Rohrer et al. However, there is another possibility that uPA activates some latent proteases311 that may participate directly
2525 in the remodeling of sclera, and PAI-1 inhibits the activation of them. Further investigation is necessary to disclose the effect of uPA and PAI-1 on the axial elongation. This study suggests that one of the retinal control on scleral growth seems to be mediated by TGF-/3 and its reduction results in the axial elongation in FDM of chick. Key Words axial length, form-deprivation myopia, plasminogen activator, plasminogen activator inhibitor, transforming growth factor-beta Acknowledgment The authors thank personnel at the Senjyu Co. Ltd. Creative Center for assisting with the treatment and measurement of animals. References 1. Wallman J, Turkel J, Trachtman J. Extreme myopia produced by modest changes in early visual experience. Science. 1978;201:1249-1251. 2. Pickett-Seltner RL, SivakJG, PasternakJJ. Experimentally induced myopia in chicks: morphometric and biochemical analysis during the first 14 days after hatching. Vision Res. 1988;28:323-328. 3. Christensen AM, Wallman J. Evidence that increased scleral growth underlies visual deprivation myopia in chicks. Invest Ophthalmol Vis Sci. 1991;32:2143-2150. 4. Wallman J. Retinal influences on sclera underlie visual deprivation myopia. In: Bock G, Widdows K, eds. Myopia and the Control of Eye Groiuth. Chiba Foundation Symposium 155. Chichester, UK: John Wiley and Sons; 1990:126-141. 5. Stone RA, Lin T, Lades AM, Iuvone M. Retinal dopamine and form-deprivation myopia. Proc Natl Acad Sci USA. 1989;86:704-706. 6. Rohrer B, Spira A, Stell WK. Apomorphine blocks form-deprivation myopia in chickens by a dopamine D2 receptor mechanism acting in retina or pigmented epithelium. Vis Neurosci. 1993; 10:447-453. 7. Ehrlich D, SattayasaiJ, ZappiaJ, Barrington M. Effects of selective neurotoxins on eye growth in the young chick. In: Bock G, Widdows K, eds. Myopia and the Control of Eye Growth. Chiba Foundation Symposium 155. Chichester, UK: John Wiley and Sons; 1990:6388. 8. Lutty G, Ikeda K, Chandler C, McLeod DS. Immunohistochemical localization of transforming growth factor-/? in human photoreceptors. Curr Eye Res. 1991; 10:61-74. 9. Tanihara H, Yoshida M, Matsumoto M, Yoshimura N. Identification of transforming growth factor-/? expressed in cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1993; 34:413-419. 10. Roberts AB, Anzano MA, Wakefield LM, et al. Type /? transforming growth factor: a bifunctional regulation of cellular growth. Proc Natl Acad Sci USA. 1985:82:119-123.
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