Collagen Gene Expression and the Altered Accumulation of Scleral Collagen during the Development of High Myopia*

Alex Gentle, Yanyan Liu, Jennifer E. Martin, Giada L. Conti, and Neville A. McBrienDagger

From the Department of Optometry and Vision Sciences, The University of Melbourne, Victoria 3010, Australia

Received for publication, January 29, 2003, and in revised form, February 25, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The development of high myopia is associated with reduced scleral collagen accumulation, scleral thinning, and loss of scleral tissue, in both humans and animal models. Reduced collagen fibril diameter is also observed in the sclera of eyes with high myopia. The present study investigated aspects of scleral collagen synthesis and degradation, in a mammalian model of high myopia, to elucidate the factors underlying scleral changes. General synthesis and degradation of scleral collagen was investigated in monocularly deprived tree shrews, through the in vivo administration of [3H]proline and subsequent assay of scleral tissue for [3H]collagen. In addition, PCR enriched cDNA, produced from tree shrew scleral mRNA, was used to synthesize probes for hybridization to custom gene arrays consisting of partial sequences for 11 collagen subtypes. Finally, real-time reverse transcriptase-PCR was employed to investigate collagen type I, III, and V mRNA expression in the sclera of myopic, contralateral control, and normal tree shrew eyes. Scleral [3H]proline incorporation was reduced at the posterior pole of myopic eyes following 5 days of monocular deprivation (-36 ± 4%), whereas [3H]proline content was similar in treated and control eyes before myopia induction (-1 ± 8%) but was reduced in myopic eyes following 5 (-8 ± 2%), 12 (-15 ± 4%), and 24 (-10 ± 4%) days of myopia induction. The majority of the collagens investigated were found to be expressed in the sclera, with 11 subtypes being identified. Collagen type I mRNA expression was reduced in the sclera of myopic eyes (-20 ± 7%), however, collagen type III (+2 ± 9%) and type V (-1 ± 6%) expression was unchanged relative to control, resulting in a net increase in the ratio of expression of collagen type III/type I and collagen type V/type I (22 and 25%, respectively). These results show that reduced scleral collagen accumulation in myopic eyes is a result of both decreased collagen synthesis and accelerated collagen degradation. Furthermore, changes in collagen synthesis are driven by reduced type I collagen production. Short term increases in the ratio of newly synthesized collagen type III/type I and type V/type I are likely to be important in the increasing frequency of small diameter scleral collagen fibrils observed in high myopia and may be important in the subsequent development of posterior staphyloma in humans with pathological myopia.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The refractive power of the eye is correlated with ocular axial length and it is well established that myopia is caused by increased axial eye size (1). Studies, in humans and animal models, have shown how this increased axial length is predominantly a consequence of increased vitreous chamber depth, rather than marked changes in any other ocular component parameter such as anterior chamber depth or lens thickness (2, 3). The prevalence of human high myopia (usually defined as eyes with >6 dioptres (D) of myopia, or >26 mm in length) is ~2% in the general population (1) and it is well documented that individuals with high myopia have a greatly increased risk of ocular pathology (4). Current thinking holds that excessive elongation of the vitreous chamber is the causative factor in the development of chorioretinal pathology, likely because of the increased biomechanical stresses that are placed on the retina and choroid in an enlarged eye.

The development of high myopia in humans is associated with marked thinning of the sclera, the tough outer coat of the eye that facilitates any change in eye size. Scleral thinning is greatest at the posterior pole of the eye, the anatomical region of greatest retinal photoreceptor density and vital to detailed visual discrimination (5, 6). In some individuals, the thinned posterior sclera precipitates local ectasic change or staphyloma (7, 8). This altered scleral morphology is associated with local changes in collagen fibril ultrastructure and increased numbers of small diameter collagen fibrils. In addition, there is a more lamellar organization of posterior scleral collagen fibril bundles (7, 9). Similar characteristics are reported in the sclera of mammalian models of myopia progression, such as the tree shrew, where scleral thinning and tissue loss precede the appearance of more small diameter collagen fibrils and altered fibril bundle morphology (10).

Collagen accounts for 90% of scleral dry weight, the majority of this being collagen type I (11, 12). Mammalian sclera also contains small amounts of other fibrillar and fibril-associated collagens (13-15) and studies have shown that scleral fibrils are heterologous, comprising collagen types I, III, and V (13). To date, at least 21 different collagen subtypes have been identified (16), with subtype expression patterns tissue- and structure-specific. As a result, it is likely that additional collagen subtypes to those already identified are expressed in the mammalian sclera. Of particular note is the fact that in tissues such as the cornea, minor fibrillar collagen subtypes, particularly collagen type V, are important in regulating lateral accretion of collagen, thus controlling fibril diameter (17).

Mammalian models of myopia have shown that scleral thinning and tissue loss occur rapidly in myopia development (10). Scleral tissue loss occurs in conjunction with increased expression and activation of collagen-degrading enzymes, such as matrix metalloproteinase-2 (18), decreased production of glycosaminoglycans (19, 20) and decreased proliferative activity of scleral cells (21). The net result is a decrease in scleral collagen content (22), as also found in human high myopia (23). However, there has to date been no explicit demonstration of the process of collagen degradation in eyes with progressive myopia. Scleral remodeling accompanies alteration in its material properties, with the sclera becoming more extensible in myopic eyes. Studies show that increased extensibility is not solely accounted for by scleral thinning, suggesting that altered scleral biochemistry results in changes to the physical properties of the matrix (24, 25). The majority of scleral thinning and tissue loss occur well before the increased frequency of smaller collagen fibrils (10), suggesting that changes in fibril diameter are secondary to the initial process of scleral thinning in myopic eyes. However, given that collagen turns over relatively slowly in mature tissues (26), it is likely that the immediate effects of altered scleral biochemistry on collagen fibril morphology do not manifest for some time.

This study investigated two important issues relating to the role of scleral collagen in the development of high myopia, using the well characterized tree shrew model of myopia (27). First, it was established whether general collagen synthesis, collagen degradation, or a combination of the two, underlies reduced scleral collagen accumulation and tissue loss in eyes with progressive myopia. Second, expression of a range of collagen subtypes was demonstrated in the sclera, before short term expression patterns of collagen subtypes III and V, in conjunction with those of type I, were investigated in myopic eyes, to determine their possible role in fibril diameter changes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Experimental Subjects and in Vivo Procedures-- Tree shrew (Tupaia belangeri) pups were maternally reared in our breeding colony before being transferred to individual cages and assigned to one of the experimental groups. Following the commencement of experimental procedures, animals were maintained on a 15/9 h light/dark cycle and food and water were available ad libitum. Myopia was induced monocularly, using a translucent occluder attached to a head-mounted durilium goggle, which was fitted to animals under anesthesia (ketamine, 90 mg/kg; xylazine, 10 mg/kg) using the procedure previously described (28). The unoccluded eye served as a paired control. The myopia-inducing goggle was always fitted 15 days after natural eye-opening, the start of the period where tree shrews have been found to be most susceptible to the inducement of myopia (27, 29). Numbers of right or left eye treatments were as balanced as possible within each group. All of the procedures were carried out in accordance with the National Health and Medical Research Council of Australia's Guidelines for the Care and Use of Animals in Research.

General scleral collagen production was investigated in one group of animals (n = 6) that had myopia induced for 5 days before newly synthesized collagen was labeled via delivery of a radiolabeled collagen precursor ([3H]proline) and the scleral tissue was collected and assayed. Four groups of animals (n = 5 each group) were used to investigate the elimination of collagen from the sclera. Animals were injected with the radiolabeled collagen precursor, allowed a suitable period for collagen labeling (24 h), then scleral tissue was collected (time 0) or myopia was induced for periods of 5, 12, or 24 days before the tissue was collected. Scleral tissue was then assayed for the amount of labeled collagen remaining in the sclera.

One group of untreated animals (n = 2) was used to determine collagen expression profiles in the sclera. Whole scleral tissue was collected from these animals 20 days after natural eye opening, representing an equivalent age to the animals that underwent 5 days of myopia induction. Scleral collagen mRNA expression in myopia was investigated in one group of animals that had myopia induced for 5 days (n = 8), whereas one group of age-matched untreated animals (n = 4) were employed as a control for this study.

Ocular axial dimensions and ocular refraction were measured at the end of the treatment period in all animals, using A-scan ultrasonography and streak retinoscopy, respectively, as has previously been reported (30). Measurements were taken after the animals had been placed under anesthesia (ketamine, 90 mg/kg; xylazine, 10 mg/kg). Following ocular measurements, eyes were enucleated with animals under terminal anesthesia (120 mg/kg sodium pentobarbital).

In animals used to investigate general collagen synthesis and degradation, both eyes were enucleated and the left eye was dissected before the right eye, thus randomizing whether the treated or control eye was dissected first. Following enucleation, extraneous orbital tissue was cleaned away and the anterior structures were removed with scissors cutting close to the limbus. The posterior segment was flat-mounted and a 7-mm trephine, centered on the posterior pole of the eye, was used to separate the sample into posterior and anterior/equatorial samples. These samples were cleaned of retina and choroid. The optic nerve head was removed from the posterior sample using a 1.5-mm trephine. The lens and ciliary body were removed from the anterior ocular structures and a 5-mm trephine was used to isolate the central cornea for use as a control. The corneal stroma is a collagenous extracellular matrix that is continuous with the sclera but does not alter its rate of matrix turnover during myopia progression (19). All samples were snap frozen in liquid nitrogen and then stored at -80 °C until assayed. In animals utilized for mRNA expression studies, the dissection process was essentially identical, except the fact that the left eye was removed and dissected while the animal was maintained under deep anesthesia on a heating pad, following that the right eye was removed and dissected. The scleral samples were the only tissues collected from these animals and all samples were snap frozen in liquid nitrogen within 6 min of enucleation.

Delivery of [3H]Proline-- In the group of animals that provided information regarding collagen production, an intraperitoneal injection of 300 µCi of L-[2,3,4,5-3H]proline (1 mCi/ml, 3.7 TBq/mmol; PerkinElmer Life Sciences) was administered on the final morning of the procedure. Proline is a relatively specific collagen precursor, accounting for ~4% of non-collagenous proteins and 14% of collagen (31). However, when proline is assayed in a tissue such as the sclera, which comprises around 90% collagen (11), almost all of that assayed will represent collagen. A period of 9 h was allowed for incorporation of the label into newly synthesized collagen before ocular measurements were taken and the tissue was collected for assay. Pilot studies suggested that peak label incorporation was reached by this point. In the four groups of animals that provided information of general collagen degradation, an intraperitoneal injection of 300 µCi of L-[2,3,4,5-3H]proline was delivered 24 h prior to the onset of monocular occlusion, the literature suggesting that even in a rapidly remodeling tissue such as the neonatal cornea, 24 h is sufficient for peak incorporation of label incorporation to have passed (32). At the time of fitting of the occluder, a dose of "cold" proline (7 mmol/kg L-proline in 0.9% saline) was administered, the dose being 100 times that of the radiolabeled proline, with the intention of diluting intracellular proline precursor pools and limiting the potential for reutilization of label (33).

Materials-- MultiScreen filtration plates (0.65 µm pore size, Durapore membrane, number MANP N6510) were obtained from Millipore (Bedford, MA); cytoscint liquid scintillation fluid from ICN (Irvine, CA); pepsin, L-hydroxyproline, chloramine T, and p-dimethylaminobenzaldehyde were from Sigma; Macro arrays were custom made to our design by the Australian Genome Research Foundation (AGRF, Melbourne, Australia); SMART cDNA synthesis kit and the Advantage 2 PCR kit were obtained from Clontech (Palo Alto, CA); Micro Bio-Spin 30 columns were obtained from Bio-Rad; [alpha -32P]dCTPs, Rapid Hyb buffer, and Megaprime labeling kits were obtained from Amersham Biosciences; PCR primers were obtained from Genset (South La Jolla, CA); guanidine thiocyanate, DNase I, Moloney murine leukemia virus reverse transcriptase, dNTPs, and RNasin were obtained from Promega; FastStart DNA Master Mix was obtained from Roche Molecular Biochemicals (Mannheim, Germany); QIAquick PCR purification kits were obtained from Qiagen (Valencia, CA); CEQ DTCS Quick Start sequencing kits were obtained from Beckman; All other reagents were purchased from Sigma.

Assay of Radiolabeled Collagen-- Tissue was homogenized in sterile de-ionized water using a freezer mill. The homogenate was then mixed with pepsin and acetic acid to give final concentrations of 2 mg/ml and 0.5 M, respectively. The samples were gently agitated at 4 °C for 48 h. The digested samples were then centrifuged to pellet insoluble material and the supernatant was collected for assay. Connective tissues contain collagen fractions that are variously soluble under specific conditions. Our pilot studies and previous reports indicated that the above method gave the best recovery of collagen for minimal processing (34), however, to control for variations in extraction efficiency, raw data were always normalized to hydroxyproline content of the homogenate.

The [3H]proline assay was adapted from a previous report with slight modifications to suit this experiment (35). Filters of the multiwell plate were wetted with 100 µl of 25% trichloroacetic acid. Triplicates (100 µl) of each corneal, anterior/equatorial, or posterior scleral sample were added to the wells with 100 µl of 50% trichloroacetic acid. The plates were then incubated at 4 °C for 1 h with gentle agitation to precipitate the macromolecules. The precipitate was collected on the filter membrane by draining thoroughly on a vacuum manifold, then washed 3 times with 300 µl of 10% trichloroacetic acid to remove the unincorporated label. The filters were allowed to air dry before being punched into plastic scintillation vials containing 500 µl of 4 M guanidine hydrochloride in 33% isopropyl alcohol. The vials were incubated overnight with gentle agitation to dissociate the collagen from the filter. The radiolabel was quantified through the addition of 10 ml of cytoscint and scintillation counting. Data (disintegrations/min) were corrected for isotope counting efficiency using the internal standard and isotope library of the machine.

Hydroxyproline content, a highly specific indicator of collagen content in each sample, was determined using a previously described assay (36). Standards of hydroxyproline were prepared (0.5-8.0 µg in 40 µl) and 10 µl of sodium hydroxide (10 M) was mixed with 40 µl of sample or standard in autoclavable tubes. Each sample and standard was hydrolyzed by autoclaving at 120 °C for 20 min. Chloramine-T solution (0.056 M in 10% 1-propanol and acetate citrate buffer, 450 µl) was added to each hydrolysate after cooling and, after mixing gently, the oxidation reaction proceeded for 25 min at room temperature. Ehrlich's aldehyde reagent (1 M p-dimethylaminobenzaldehyde in 1-propanol/perchloric acid, 2:1, v/v) was freshly prepared and added to each vial (500 µl) and mixed gently. All samples and standards were then incubated in a water bath at 65 °C for 20 min to develop the color change. The color density was quantified at 550 nm using a spectrophotometer.

Collagen Gene Array Design and Validation-- The 40,000 Human Unigene clone library at the AGRF was searched to find available IMAGE clone sequences for the various collagen subtypes. Where multiple alpha -subunit clones were available for a given collagen subtype, the most common naturally occurring alpha -subunit, usually alpha 1, was selected. The sequence associated with the accession number of each selected clone was confirmed using the BLAST facility at the NCBI (www.ncbi.nlm.nih.gov:80/BLAST). Once clones had been validated (subunits representing collagens I-IX, XI, and XIII-XVIII) the custom array was produced and included two ubiquitously expressed genes as positive controls HPRT1 and GAPDH. The final array had each selected clone represented in duplicate. Colonies were grown, transferred onto membranes, and lysed at the AGRF. The colony plates were supplied with the arrays to allow sequence validation. Following delivery of the arrays, it was found that the colonies containing COL2A1 and COL14A1 had not grown and further investigation, through colony PCR, gel electrophoresis, and sequencing with the Beckman Coulter CEQ 8000, revealed that "COL4A1," "COL5A2," and "COL11A1" were not the sequences anticipated. As a result of these findings, positive hybridization signals to any of these positions on the array could not be considered to represent the collagen subtype of interest.

Total RNA Extraction-- Tissues were homogenized (4 M guanidine thiocyanate, 25 mM sodium citrate) in a freezer mill, then total RNA was isolated using a standard method (37). Phenol:chloroform reagent (Sigma number P1944) was added to the samples, RNA was precipitated from the aqueous phase using isopropyl alcohol, then the resultant pellet was washed twice with 70% ethanol. Glycogen (10 µg) was added prior to isopropyl alcohol precipitation to maximize RNA recovery. The resuspended pellet was protected with RNasin and treated with DNase I to remove potential genomic DNA contamination. The RNA was repurified as before. The RNA content of the resuspended pellet was quantified and checked for purity and condition by spectrophotometry and gel electrophoresis.

Reverse Transcription-- SMART cDNA synthesis was carried out using 1 µg of total RNA isolated from whole sclera. SMART technology produces cDNA libraries with common primer sites, thus allowing amplification of the library via PCR. The reaction was carried out using the reagents, and in accordance with the protocol, supplied with the kit. The cDNA library generated was then enriched through PCR with the Advantage 2 PCR system and the SMART primer. PCR products were monitored through agarose gel electrophoresis and 30 cycles of PCR was found to be optimal for the amplification of this library. PCR products were purified using the QIAquick PCR purification kit, quantified, and stored for use as probe templates.

Posterior scleral sample mRNA from myopic and normal animals was reverse transcribed using a standard technique. Amounts of 100-500 ng of total RNA were used depending on the amount recovered from each eye. Identical amounts of RNA were always reverse transcribed from scleral RNA samples of paired treated and control eyes. Standard reverse transcriptase reactions for paired samples were carried out from the same reagent master mix (containing Moloney murine leukemia virus reverse transcriptase, RNasin, and dNTPs) and at the same time.

Gene Array Hybridization-- Radiolabeled probes were prepared from 100 ng of the PCR-enriched cDNA library using the Megaprime DNA labeling system, [alpha -32P]dCTPs, and the protocol supplied. The synthesized probes were purified by application to Micro Bio-spin P-30 columns, checked for activity by scintillation counting, and stored at -20 °C before use. Using a hybridization oven, macroarrays were prehybridized for 30 min at 45 °C in Rapid Hyb buffer, then the probes were denatured and added to the hybridization bottle at 68 °C. The hybridization was carried out for 24 h and was followed by one wash at room temperature (20 min), then two washes at 68 °C (15 min) in 2× SSC, 0.1% SDS. The array was visualized using a PhosphorImager.

Gene array hybridizations were screened using an RT-PCR and sequencing protocol developed in this laboratory (38). Briefly, primers were designed against areas of the coding regions of COL1A1, COL2A1, COL3A1, COL5A1, and COL8A1 that were found to be well conserved across species. Each primer was also checked to ensure there was little or no cross-subtype specificity. These primers were then used to perform PCR reactions on scleral SMART cDNA and amplified fragments were purified and sequenced to confirm their identity.

Collagen mRNA Expression-- Scleral tissue samples were processed for gene expression analysis using real-time PCR. Data was collected from treated and control eyes of 8 myopic and 4 age-matched normal animals. Semiquantitative real-time PCR was carried in paired treated and control or right and left eyes for COL1A1, COL3A1, and COL5A1, and HPRT, a housekeeping gene whose expression was found not to be changed during the development of, or recovery from, myopia (39). Tree shrew sequence-specific primers were generated from the sequence information obtained in previous experiments. Final primer sequences and product sizes are given in Table I. The PCR assays were carried out in triplicate to improve the accuracy of estimates. Templates (2-4 µl) were mixed with primers (1 µM) and the FastStart DNA master mixture containing FastStart Taq polymerase, SYBR green I dye, dNTPs, and PCR buffer. Additional magnesium chloride was added as appropriate. Reactions were carried out in the LightCycler (Roche Molecular Biochemicals) real-time PCR machine. Standard cycling conditions for this machine were used (10 min initial denaturation at 95-97 °C, then 40 cycles of denaturation 95-97 °C for 0-10 s, annealing at 58-60 °C for 10 s, extension at 72 °C for 8-20 s, dependent on product size) following which detection was carried out. Melting curves were then used to ensure the purity of the amplified product.


                              
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Table I
Tree shrew-specific primer sequences and PCR product sizes

Data Analysis-- Ocular biometric data was presented either as absolute values or as the mean interocular difference between eyes (treated-control) ± S.E. Values of [3H]proline incorporation (disintegrations/min) were normalized to hydroxyproline content (dpm/µg) and either analyzed as absolute values or as the mean percentage difference between eyes ((treated-control)/control) ± S.E. Comparisons were made between the mean of triplicate PCR cycle curves of treated and control eyes, in the log linear phase of the PCR reaction, using GraphPad Prism software (GraphPad Software Inc., San Diego, CA) as previously reported (38). Fold difference between eyes was normalized to that of the housekeeping gene, then converted to a percentage difference. Data are presented as the mean percentage difference ± S.E. Statistical comparisons were performed either using a paired t test or a general linear model ANOVA with Tukey's post-hoc analysis for comparison across groups. All analyses was performed on Minitab software (Minitab Inc. State College, PA), unless stated.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ocular Biometry and Refraction-- In the two groups of myopic animals (n = 14) used to investigate general and subtype-specific collagen production, 5 days of monocular deprivation produced -8.7 ± 0.9 dioptres (p < 0.001) of relative myopia between the treated and control eyes. This resulted from a relative increase in ocular axial length of 0.18 ± 0.02 mm (p < 0.001), which was predominantly caused by a vitreous chamber depth increase of 0.16 ± 0.02 mm (p < 0.001) in the treated eye. No other ocular parameter altered significantly between myopic and control eyes, as has previously been reported (19).

In animal groups used to evaluate collagen degradation, after the statistical method had allowed for processing order effects, there was a significant treatment effect on refractive error (F (3,15) = 178.7, p < 0.001), vitreous chamber depth (F (3,15) = 88.3, p < 0.001), and axial length (F (3,15) = 76.2, p < 0.001), as expected. No significant changes were found in any other ocular parameter over the treatment period (Table II). In addition to the treatment effects, control eye data shows there is a general increase in lens thickness and axial length with age, which was also as expected (27).


                              
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Table II
Ocular biometry in tree shrews during the development of induced axial myopia
Absolute values represent the group means of treated and control or right and left eyes ± SE. Interocular differences are expressed as the group mean of the differences between treated and control or right and left eyes ± SE.

General Collagen Synthesis-- The mean hydroxyproline level in posterior scleral extracts of control eyes in the present study was 59.7 ± 3.5 µg, which we estimate represents ~40% of the collagen present, assuming a collagen hydroxyproline content of 10% and a scleral collagen content of 90% (11, 40). This finding is in keeping with a previous report in corneal tissue (34). Similar extraction efficiencies were obtained for both corneal (48.0 ± 4.0 µg) and anterior/equatorial scleral samples (65.1 ± 5.4 µg). There was found to be no significant difference in the amount of hydroxyproline extracted from paired treated or control eyes for any tissue. Labeled proline incorporation (dpm/µg) into newly synthesized collagen was reduced in the posterior sclera (-36 ± 4%, p < 0.001 paired t test) and total sclera (-28 ± 5%, p < 0.01) after 5 days of myopia induction (Fig. 1). No statistical difference in proline incorporation was found in the anterior/equatorial sclera or the cornea of the same eyes (Fig. 1). There was no significant correlation between the difference in axial eye size and the magnitude of change in proline incorporation in these animals.


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Fig. 1.   Collagen synthesis, as estimated by the interocular difference in [3H]proline content, is significantly reduced in the posterior and total sclera, but not significantly altered in the anterior/equatorial sclera or cornea, of young tree shrews following 5 days of myopia development. n = 6. **, p < 0.01; *, p < 0.05.

General Collagen Degradation-- After statistical analysis had accounted for processing order effects there was a statistically significant treatment effect in the posterior sclera of treated eyes (F (3,16) = 7.1 p < 0.001), demonstrating that the elimination of labeled proline (dpm/µg) from eyes developing myopia was more rapid than from contralateral control eyes. Furthermore, pairwise comparison showed there to be a significant interocular difference between the 12-day MD group and the time 0 control group (p < 0.05), however, this was not significant in other groups, despite the fact that a paired t test showed there to be a significant difference between treated and control eyes at 5 (-8 ± 2%, p < 0.05) and 12 (-15 ± 4%, p < 0.05) days (Fig. 2A). In addition, there was a significant overall treatment effect in the total sclera of the treated groups (F (3,16) = 5.3, p < 0.01), suggesting there was also a more rapid elimination of proline from the whole sclera of eyes developing myopia, although the pairwise comparisons between the 5-, 12-, and 24-day MD groups and the time 0 control group were not significant (p = 0.08; Fig. 2B). No significant changes were found with time in the treated eye of the anterior sclera alone or of the cornea.


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Fig. 2.   A, posterior scleral collagen degradation is significantly increased in eyes developing myopia, as indicated by general linear model ANOVA (p < 0.001). The graph shows the percentage difference in [3H]proline content between treated and control eyes of animals monocularly deprived for 0, 5, 12, or 24 days. B, total scleral collagen degradation is significantly increased in eyes developing myopia, as indicated by general linear model ANOVA (p < 0.01). The graph shows the percentage difference in [3H]proline content between treated and control eyes of animals monocularly deprived for 0, 5, 12, or 24 days. n = 5 each group. *, p < 0.05 by ANOVA pairwise comparison.

Collagen Gene Array Findings-- Positive hybridizations were obtained for 9 of the 11 validated collagen alpha -subunits investigated using the macroarray (Fig. 3, A and B), the two subunits not hybridizing being COL8A1 and COL18A1. The ubiquitously expressed genes HPRT and GAPDH showed positive hybridization signals, as expected.


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Fig. 3.   A, phosphorimage of representative collagen gene array following hybridization with radiolabeled SMART cDNA probes, generated from scleral mRNA. Clones containing specific collagen subtype fragments and validated by sequencing are indicated. Duplicates of each clone were present, related clones being indicated using lines. The ovals indicate the positive hybridizations where sequencing had shown the clone sequences were not collagen subtypes. B, the tabulated data indicates the collagen subtypes that were to be investigated, the accession numbers of the clones immobilized on the array, clone sequence verification data, whether a positive hybridization was found on both membranes, if the array result was confirmed by PCR, and sequencing and whether a particular collagen subtype was found to be present in the sclera. Check mark indicates an affirmative response to the question stated as the column title, cross indicates a negative response, question mark indicates that from the data collected it was not possible to give either an affirmative or negative response, and dash indicates that no data was collected in this respect.

Of the selected collagen subtypes that were screened using RT-PCR and sequencing, COL1A1 and COL3A1 were found to be present in the sclera, thus validating the positive array finding in this respect. However, COL8A1 was also found to be present in the sclera, despite the fact that it did not hybridize to the array, suggesting that the technique may generate false negative results. In addition, of the collagen species not represented on the array, COL5A1 was found in the sclera, whereas COL2A1 was not present (Fig. 4). The gene array findings were replicated in whole sclera from a second tree shrew, thus demonstrating the robustness of the technique in this respect. The results are summarized in Fig. 3, A and B, and show that the mammalian sclera expresses message for at least 11 collagen subtypes.


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Fig. 4.   Gel electrophoresis of PCR products following amplification with primers to collagen type II. The first lane shows no amplification from tree shrew scleral SMART cDNA, whereas a product of the expected size was obtained from tree shrew ear cartilage cDNA (second lane). The product shown in the second lane was sequenced and its identity confirmed as type II collagen.

Collagen Subtype mRNA Expression-- Analysis of collagen gene expression, normalized to the expression of the housekeeping gene HPRT, showed there to be a significant decrease in posterior scleral collagen type I mRNA expression in the myopic, relative to the contralateral control, eye (-20 ± 7%, p < 0.05) as previously reported for the whole sclera (Fig. 5A). However, the mRNA expression patterns of collagen type III (+2 ± 9%, p = 0.84) and collagen type V (-1 ± 6%, p = 0.74) were not significantly altered by 5 days of myopia induction (Fig. 5A). These findings differed from those in the normal animals assessed, where there was found to be no significant change in subtype expression (Fig. 5A). The reduction in type I collagen gene expression in myopic eyes resulted in a significant relative increase in the ratio of type III/type I (+22%) and type V/type I (+25%) collagen expression (p = 0.05; Fig. 5B). No change was observed in normal eyes when this ratio was calculated using a within animal comparison (Fig. 5B).


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Fig. 5.   A, type I collagen mRNA expression is reduced in the posterior sclera of myopic, relative to contralateral control, eyes following 5 days of myopia development. Expression levels of types III and V collagen were found to be unaltered relative to control. No significant changes were found in age matched normal eyes for any of the subtypes investigated. n = 8 in the myopic group and n = 4 in the normal group. *, p < 0.05 by paired t test. B, when a within animal comparison was made of the type III/type I and type V/type I collagen expression ratios, the ratio was found to be elevated in myopic, but not normal, animals. n = 8 in the myopic group and n = 4 in the normal group. n = 8 in the myopic group and n = 4 in the normal group. *, p = 0.05 by paired comparison.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Scleral thinning, tissue loss, and altered tissue morphology are associated with the development of high myopia in humans and animals models of myopia and the current study makes three major contributions to the literature in this area. First, this study shows that reduced collagen accumulation in the myopic eye results from both reduced collagen synthesis and accelerated collagen degradation in the sclera, which until now could only be speculated upon from previous findings. Second, this study shows that scleral thinning early in high myopia development is associated with differential changes in collagen subtype expression and provides the first evidence of a mechanism that may underlie the longer term changes in fibril diameter reported in the sclera of humans with pathological myopia and animal models of high myopia. Third, this study represents a comprehensive demonstration of many of the collagen subtypes for which message is expressed in the mammalian sclera.

Collagen Accumulation in Myopia-- Reduced proline incorporation in the sclera of eyes following 5 days of myopia development is consistent with the conclusion that general collagen production is reduced in the sclera of eyes developing myopia. Indeed, although proline is not exclusive to collagen in the body, previous studies have found that only 4% of non-collagenous proteins comprise proline (31). Furthermore, some 90% of the scleral dry weight is collagen (11) and the vast majority of this is fibrillar collagen type I (12), indicating that nearly all of the radiolabeled proline assayed in the sclera during the present study would have been incorporated in collagen type I. Reduced collagen synthesis in eyes developing myopia is consistent with the results of both this and a previous study (41), in that collagen type I mRNA production is reduced following different periods of myopia development. The findings of the present study confirm that reduced accumulation of collagen in the sclera of myopic eyes, as determined from the reported reduction in scleral dry weight and reduced scleral hydroxyproline content (19, 22), is partly accounted for by reduced collagen synthesis.

In addition, the current study employed a modification of the experimental paradigm to radiolabel scleral collagen, prior to the induction of myopia, to demonstrate that labeled collagen was eliminated more rapidly from the sclera of eyes in which myopia was developing. This is, to our knowledge, the first study to directly show that accelerated collagen degradation is associated with scleral thinning and tissue loss in mammalian eyes with progressive myopia. Furthermore, this is consistent with studies that show an increase in the activity of collagen degrading enzymes, such as matrix metalloproteinase-2, during myopia progression in animal models of myopia (18).

The findings of the current study show that the elimination of collagen from the posterior region of the sclera is most rapid during the first 12 days of myopia development. This is consistent with previous findings that indicate that the majority of posterior scleral tissue loss in high myopia development in this mammalian model occurs over the first 12 days (Fig. 6) (10, 19). These studies also demonstrate that the lost tissue is not replaced if myopia continues to progress (10). Indeed, when considered in the context of human high myopia, these findings imply that scleral tissue loss occurs much earlier than the clinical signs of pathological myopia, which only usually manifest after high levels of refractive error have been present for some time.


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Fig. 6.   Collagen degradation in the posterior sclera, as indicated by the interocular difference between treated and control eyes with time, follows a similar pattern to posterior scleral dry weight loss. The patterns of scleral dry weight loss are plotted for 0, 5, and 12 days of myopia development and projected out to 8 months using previously published data (10, 19).

The rapid manifestation of altered collagen accumulation in the sclera of eyes developing myopia suggests that it may contribute to the rapid alterations that have been reported in the mechanical properties of the sclera in myopic eyes (25). Such a finding is expected given the major role that collagen plays in determining the biomechanical properties of many different tissue systems (42).

Fibrillar Collagen Synthesis in Myopia-- To evaluate the role that modulation of fibrillar collagen subtype-specific gene expression plays in the long term alterations to the sclera in myopia, the present study investigated changes in the production of mRNA encoding the fibril-forming collagen types I, III, and V in posterior scleral tissue in tree shrews. Each of the three collagen subtypes has previously been shown to contribute to the scleral collagen fibrils of the sclera of other mammalian species (13), and it is widely reported that variation in fibril composition of these subtypes is critical to fibril diameter (17). Analysis of mRNA levels in the sclera of myopic, relative to contralateral control, eyes showed a significant reduction in collagen type I mRNA expression, as previously reported (41). Importantly, however, there was no evidence of a similar reduction in the expression of collagen type III and type V. Indeed, unlike collagen type I, no significant difference in mRNA expression was observed between treated and fellow control eyes for these quantitatively minor, but important components of the scleral collagen fibrils.

The differential expression patterns observed for the fibril-forming collagens investigated demonstrate that the relative ratio of collagen type III/type I and collagen type V/type I mRNA is increased in the sclera of eyes with progressive myopia, with significant increases of some 22 and 25% in these respective ratios. This relative change in the ratio of collagen subtype production occurs during one of the most rapid phases of collagen synthesis and tissue remodeling in the developing tree shrew eye. Smaller diameter collagen fibrils have been observed in the sclera of highly myopic human (9) and monkey eyes (43) and the accumulation of a significant number of small diameter collagen fibrils has also been reported in the sclera of highly myopic tree shrew eyes following relatively long (3 to 9 months) periods of myopia development (10). Studies in other connective tissue systems have examined the role co-assembly of different collagen types plays in regulating fibrillogenesis and such studies have provided evidence to suggest that fibril diameter is modulated according to the specific composition of the fibrils (17). The effects of collagen type I/type V interactions on fibril diameter regulation have been best demonstrated using in vitro self-assembly assays, where the presence of increasing amounts of collagen type V was found to result in decreased fibril diameters (17). Of particular note is the fact that for a relative increase in collagen type V of 20%, a value comparable with the relative increase in the type V/type I ratio observed in the present study, there was a relative decrease in collagen fibril diameter of 36% (relative to homotypic collagen type I fibrils), which is consistent with the approximate 34% decrease observed in the collagen fibril diameters of the outer posterior scleral "layers" of myopic tree shrew eyes (10). It is hypothesized that larger diameter collagen fibrils develop through lateral accumulation of molecular collagen on small diameter immature fibrils, and that the presence of collagen type V inhibits this lateral accumulation. Indeed, studies in the corneal stroma, an ocular tissue closely associated with the sclera and containing particularly high concentrations of type V collagen, suggest it is this collagen subtype that is responsible for the small and regular collagen fibrils that are characteristic of this tissue (17).

We propose that the increase in expression of fibrillar collagen type V, and perhaps collagen type III, which is also associated with reduced fibril diameter (44), relative to that of type I collagen in the present study, is indicative of a similar mechanism of fibril diameter control in the sclera of highly myopic tree shrew eyes. Inhibition of the lateral growth of newly synthesized fibrils during this early stage would potentially lead to the gradual accumulation and predominance of the smaller diameter fibrils, observed in long-term myopia. Given that this observed alteration in collagen expression is associated with a period of rapid eye growth (27), it seems likely that even a relatively short period of aberrant collagen synthesis might have profound effects on the fibril diameter distributions observed later in axial myopia development. Indeed, data from a number of connective tissue systems suggests that collagen residency times are protracted in the body (26). This may explain why the impact of early changes in fibril constituents may not be apparent until later in development. Presumably, a continued predominance of small diameter fibrils would require that collagen synthesis be altered to some extent over a longer time period and this remains to be demonstrated.

Collagen Subtype Expression in the Sclera-- The present study demonstrates that the modified gene array approach reported can be highly specific in identifying mRNA sequences of known genes in novel species. This is evidenced by the fact that of the positive array hybridizations that were screened using RT-PCR (collagen types I and III), each subtype was confirmed as present in the sclera. However, findings also suggest that the technique has a limited capacity to identify a negative result as evidenced by the finding that collagen type VIII did not hybridize to the array but was found to be present in the scleral cDNA when screened using RT-PCR. Collagen type II was not found to be present in the mammalian sclera, however, problems with the array production in respect of this clone prevent us from commenting on the likelihood of false positive hybridizations. We conclude that this array approach represents a useful tool in the identification of mRNA populations in novel species.

Data from the current study serves to demonstrate that the collagen subtype expression profile in the mammalian sclera is wider than reported to date. Collagen subtypes I, III, V, VI, VIII, and XIII were found to be present in the tree shrew sclera, as expected given previous reports of their presence in the human sclera (13, 45, 46). In addition, collagen subtypes VII, IX, XV, XVI, and XVII were identified in the tree shrew sclera and these are also likely to be present in the human sclera. RT-PCR and sequencing results suggest that collagen type II is absent from the tree shrew sclera and, although type II collagen was recently identified in the adult rodent sclera (47), this is consistent with a previous report of its absence from the human sclera (13), suggesting that the tree shrew sclera is much closer in structure to primates than that of rodents.

In summary, the current study is the first to demonstrate that reduced collagen accumulation in the sclera of highly myopic eyes is a result of reduced collagen synthesis and increased collagen degradation. These changes occur very early on in the process of myopia development. In addition this study has shown that previous reports of reduced fibril diameter in long term myopic eyes are linked to differential expression patterns between the quantitatively major and the more minor fibrillar collagens of the sclera, during the early stages of myopia development. Finally, the current study represents a comprehensive demonstration of the collagen subtypes expressed in the mammalian sclera.

    FOOTNOTES

* This work was supported by National Health and Medical Research Council of Australia Grant 145700 and Australian Research Council Grant S0005254.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Optometry and Vision, Sciences, The University of Melbourne, Victoria 3010, Australia. Tel.: 61-3-8344-7001; Fax: 61-3-9349-7474; E-mail: n.mcbrien@optometry.unimelb.edu.au.

Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M300970200

    ABBREVIATIONS

The abbreviations used are: HPRT, hypoxanthine-guanine phosphoribosyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcriptase; ANOVA, analysis of variance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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