|Molecular Vision 2007;
Received 18 July 2007 | Accepted 2 October 2007 | Published 4 October 2007
Selective modulation of scleral proteoglycan mRNA levels during minus lens compensation and recovery
John Thomas Siegwart Jr,
Christianne E. Strang
Department of Vision Sciences, School of Optometry, The University of Alabama at Birmingham, Birmingham, AL
Correspondence to: John T. Siegwart Jr., 924 18th Street South, Worrell Building, Rm 302, Birmingham, AL, 35294; Phone: (205) 934-6733; FAX: (205) 934-5725; email: email@example.com
Purpose: To better characterize the role of proteoglycans in scleral tissue remodeling during the development of minus lens induced myopia and during recovery in tree shrews.
Methods: Competitive reverse-transcription polymerase chain reaction (RT-PCR) was used to quantify the scleral mRNA levels for aggrecan, decorin, biglycan, and lumican in a group of tree shrews following four days of monocular -5 D lens treatment (n=5) and in a group after two days of recovery after 11 days of -5 D lens wear (n=5). Values were compared with age-matched normal animals (n=5). Aggrecan was localized within the sclera using immunohistochemistry.
Results: Four days of -5 D lens wear produced axial (vitreous chamber) elongation and a myopic shift in the treated eyes. Two days of recovery produced significant refractive recovery. Aggrecan mRNA levels showed differential, bidirectional regulation. Levels in the treated eye sclera relative to the control eye were 30.8%±2.4% lower after four days of -5 D lens treatment and 51.4%±2.4% higher after two days of recovery. Decorin, biglycan, and lumican mRNA levels showed little differential regulation. However, biglycan and lumican along with aggrecan showed binocular regulation (treated and control eye mRNA levels significantly lower than normal eye mRNA levels after 4 days of -5D lens treatment). Immunohistochemical results showed that aggrecan is present in tree shrew sclera and that it is located primarily between the collagen lamella and near the fibroblasts.
Conclusions: These data suggest that the expression of aggrecan is strongly differentially modulated in the sclera during experimentally induced myopia and recovery. The modulation of aggrecan in concert with previously described changes in type I collagen and hyaluronan may play a key functional role in modulating the ability of the lamellae to slip across one another. This may be manifested in the scleral creep rate, which in turn modulates axial elongation rate and refractive state.
Visual guidance of eye growth during postnatal development has been demonstrated in a variety of vertebrate species [1-5]. When a minus (concave) lens is placed over the eye, the rate of axial elongation rapidly increases and remains elevated until the increase in axial length "compensates" for the increase in focal length produced by the minus lens [6-8]. The rate then returns to normal to maintain the renewed match of the retina to the shifted focal plane. When the lens is subsequently removed, the eye is myopic and the axial elongation rate decreases below normal levels causing the eye to "recover" back toward emmetropia as normal maturation reduces the optical power of the eye [8-10]. These experimental manipulations are thought to be a demonstration of the normal visually-guided emmetropization mechanism that establishes and then maintains emmetropia by modulating the axial growth of the eye during postnatal development. Much of myopia research is aimed at understanding this emmetropization mechanism and why it sometimes fails, resulting in refractive error.
The sclera is a key structure in the control of the axial length of the eye and, therefore, the relationship of the retina to the focal plane. In tree shrews, as in primates, the sclera is a fibrous extracellular matrix (ECM) comprised of collagens (type I collagen with much lower amounts of type III and type V), elastin, proteoglycans, and other components that are arranged in interwoven layers (lamellae) produced by scleral fibroblasts [11-13]. Experimental modulation of the axial elongation rate in animal models and presumably normal emmetropization involves modulation of scleral tissue remodeling [14-18]. Tissue remodeling is a complex process that involves the regulation of numerous gene products such as collagen and proteoglycans , matrix metalloproteinases (MMPs) that degrade the ECM , and tissue inhibitors of metalloproteinases (TIMPs) that bind to and inhibit the activity of the MMPs. In addition, TIMP-2 plays a critical role in the activation of MMP-2.
In tree shrews, scleral remodeling in eyes that are developing induced myopia in response to a monocular minus lens or diffuser is characterized by decreased levels of type I collagen [12,16,21], decreased levels of sulfated and unsulfated glycosaminoglycans (GAGs) [16,22,23], and increased levels of gelatinase-A (MMP-2) . Selective changes in mRNA levels have been found for some proteins including collagen 1(I) [12,25], MMP-2, MT1-MMP, TIMP-3 , and TGFβ . However, other mRNA levels have not shown differential changes in the treated versus the fellow control eyes: MMP-3, TIMP-1, and TIMP-2 . The observed changes in scleral mRNA levels, which are temporally associated with changes in axial elongation rate, suggest that retinally derived signals modulate scleral gene expression to remodel the scleral tissue and modulate scleral creep rate.
In contrast to the all fibrous mammalian sclera, the chick sclera has an inner cartilaginous region in addition to an outer fibrous region. Increased growth of the cartilaginous layer appears to produce the increase in axial elongation during experimentally induced myopia in chicks [14,17]. In the tree shrew, remodeling of the all-fibrous sclera appears to modulate a mechanical property of the sclera (creep rate) rather than growth per se. Creep rate, a measure of the rate of slow deformation in response to a constant force (measured here as the rate of increase in the length of a strip of sclera while under constant tension), increases during minus lens wear and decreases below normal during recovery [8,28]. The modulation of scleral creep rate may be functionally important because the scleral shell is under constant tension from the force of intraocular pressure (IOP). A decrease in the ability of the sclera to withstand the force of IOP (an increase in creep rate) would result in expansion of the scleral shell and an increase in axial length.
Proteoglycans, ECM components consisting of a core protein with attached glycosaminoglycan (GAG) chains , are thought to play an important role in the mechanical properties of biological tissue . Several proteoglycans have been identified in human sclera including decorin, biglycan, and lumican , which all belong to a group known as small leucine rich proteoglycans (SLRPs), and aggrecan, a large proteoglycan typically associated with cartilage [32,33]. Based on studies in humans, aggrecan appears to be largely localized between the collagen lamellae while decorin and biglycan are more evenly dispersed throughout the lamellae . Previous studies have provided evidence that scleral proteoglycan synthesis is altered during experimentally induced myopia in chickens [34,35], tree shrews [16,23], and monkeys . These studies reported changes in the incorporation of radiolabeled sulfate into newly synthesized GAGs, which has been taken as an indication of the rate of proteoglycan (core protein) synthesis. Although sulfate incorporation can provide an estimate of overall proteoglycan synthesis, it does not distinguish between different proteoglycans unless the proteoglycans are first separated by size. In addition, the number and length of GAG chains associated with core proteins can be altered independently from changes in core-protein abundance. Mammalian sclera also contains hyaluronan (HA), an unsulfated GAG not attached to a core protein. A study that directly measured HA levels in tree shrew sclera found that HA levels were rapidly and differentially affected during myopia development and recovery .
Altering the amount or distribution of the various scleral proteoglycans along with hyaluronan  may be a factor in the changes in the scleral creep rate that occur during experimentally induced myopia and recovery [8,28] in tree shrews. In the current study we focused on mRNA levels of four proteoglycans: aggrecan, decorin, biglycan, and lumican.
Three groups of tree shrews (Tupaia glis belangeri) with five animals in each group were included in this study. One group received four days of monocular -5 D lens treatment. Previous studies show that this is the peak of the increase in scleral creep rate and increase in axial elongation rate that occurs in response to minus lens treatment in tree shrews . A second group recovered for two days without the -5 D lens following 11 days of -5 D lens treatment. Previous studies show that compensation is nearing completion by 11 days of lens wear and that after 2 days of recovery the scleral creep rate is below normal levels . A third normal group was measured 28 days (±1 days) after natural eyelid opening (days of visual experience; VE).
A lightweight goggle frame that clipped onto a dental acrylic pedestal was used to hold the -5 D lens (polymethyl methacrylate [PMMA], 12 mm diameter, 7.5 mm base curve, Conforma Contact Lenses, Norfolk, VA) in front of the eye . The pedestal was installed at 21±1 days of VE in all groups of animals, and the animals in the lens-wearing groups were allowed three days to recover from the minor surgical procedure before visual treatment had begun. The -5 D lens was randomly placed over either the right or left eye of the four-day -5 D lens-wear group and of the two-day recovery group. The fellow control eye viewed through the open goggle frame with no lens. Visual treatment was initiated by clipping the goggle frame onto the pedestal and recovery was initiated by permanently unclipping the goggle frame. The goggle was briefly removed twice each day at approximately 9:00 AM and 4:30 PM to clean the lens. During lens-cleaning, the animals were placed in a darkened nest box. To control for any systemic effect from the minor surgical procedure to install the pedestal, the normal animals also received a pedestal at 21±1 days of VE but did not wear a goggle frame.
The refractive state (spherical equivalent at the corneal plane) was measured in awake animals with an autorefractor (Nidek, Gamagori, Japan)  with no ophthalmic or systemic atropine sulfate given at any time due to concerns that atropine could alter the effect of minus lens treatment in tree shrews . The autorefractor has been found to provide accurate measures of the amount of induced myopia in tree shrews . Axial component dimensions were measured using A-scan ultrasonography as previously described . The measurements of vitreous chamber depth were made to the anterior surface of the sclera  and not the retina-vitreous interface, which could reflect a change in choroidal thickness without a change in the location of the anterior surface of the sclera . A-scan measures were made when the pedestal was installed. Refractive state and A-scan measures were made at the end of the treatment period in the four-day treatment group. In the two-day recovery group, refractive state was only measured at the end of lens treatment because A-scan measures required anesthetizing the animal, which might have affected recovery. Both refractive state and A-scan measures were made at the end of the two-day recovery period. Normal animals were measured at 28±1 days of VE. This was exactly age-matched to the four-day lens treatment group and nine days younger than the two-day recovery group.
All procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Association for Assessment and Accreditation of Laboratory and Animal Care (AAALAC) regulations for the use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
Competitive reverse-transcription polymerase chain reaction
Details of the competitive reverse-transcription polymerase chain reaction (RT-PCR) procedures used in this study have been described previously [25,26]. A brief account follows.
Tissue was collected at the end of treatment, all animals received an overdose of pentobarbital sodium and were enucleated between 10:00 AM and 11:30 AM. Whole eyes were immediately submerged at room temperature in a stabilization agent (RNALater, Ambion, Austin, TX), which prevents RNA degradation. Scleras were quickly cleaned of non-scleral tissue at room temperature in RNALater, frozen in liquid nitrogen, and kept at -80 °C until RNA was extracted.
Total RNA was extracted from individual whole scleras (SV Total RNA Isolation System; Promega, Madison, WI). RNA concentration and purity was determined by spectrophotometry at 260 nm and 280 nm.
Tree shrew-specific primers were designed for the mRNAs of decorin, biglycan, lumican, aggrecan, and 18s rRNA as described previously . Sequencing verified the identities of the PCR products. The primer sequences are given in Table 1.
Competitive RT-PCR was used to measure the levels of mRNA for aggrecan, decorin, biglycan, lumican, and 18s rRNA. Data were quantified as copies of a target gene (aggrecan, decorin, biglycan, or lumican) per copies of the housekeeping gene (18s). A detailed explanation of the RT-PCR quantification procedure can be found in previous publications [25,42].
To examine the distribution of aggrecan within the sclera, indirect immunohistochemistry with anti-aggrecan and anti-type I collagen antibodies was performed on scleral sections from a tree shrew at 47 days of VE, just 10 days more of VE than the recovery group. The eyes were removed after administration of an overdose of sodium pentobarbital, cleaned of extra ocular tissue and hemisected. Then the vitreous and retina were removed. The resulting scleral eyecup was fixed in 2% paraformaldyhde (PFA) for 2 h and cryoprotected in 30% sucrose in 0.1 M phosphate buffer (PB). Cartilage was used as a positive control for the anti-aggrecan antibody as aggrecan is known to be a major component of cartilaginous tissue . For cartilage sections, the knee joint was dissected out and fixed in 4% PFA for five days after which the cartilage was dissected away from the joint, fixed for an additional 30 min, and cryoprotected. Cryoprotected tissue was embedded in a mixture of 50% Optimal Cutting Temperature Compound (O.C.T.; VWR Scientific, West Chester, PA)/50% aquamount (Vector Laboratories, Burlingame CA), sliced into 12 μm flat cryosections, and thaw-mounted on superfrost slides (VWR Scientific, West Chester, PA). Sections were stored at -20 °C until use. Some sections were washed in 0.1 M phosphate buffered saline (PBS), incubated in Quickstain (American Master Tech Scientific Inc., Loda, CA) for 3 min, and washed three times to stain chondrocytes (cartilage control) and fibroblasts (sclera) for structural comparison to fluorescently labeled sections.
All normal sera, immunoglobins, and antisera were diluted in 0.3% Triton X-100 in PBS. Cryosections were thawed and washed in three changes of PBS, incubated at room temperature in 10% donkey normal serum for one h, and blocked with avidin and biotin blocking solutions each for 15 min. After blocking, sections were incubated with Goat-anti-Aggrecan (AF1220; against NSO-derived recombinant human aggrecan, amino acids 20-675; R&D systems, Minneapolis, MN) at a concentration of 5 μg/ml for 12-18 h at 4 °C. After incubation, slides were washed in three changes of PBS over 30 min and incubated with biotinylated donkey anti-goat (diluted 1:300; Molecular Probes, Eugene, OR) for 1 h then washed again and incubated for 1 h with avidin-conjugated Fluorescein (diluted 1:200; Molecular Probes). The slides were then washed and incubated with rabbit-anti-collagen I (placental collagen type I; 70-XR90; Fitzgerald Industries, Boston MA) at a concentration of 2 μg/ml for 12-18 h at 4 °C then washed and incubated with rhodamine conjugated donkey-anti-rabbit. Specificity controls included omission of the primary antibodies and substitution of the same protein concentration of goat or rabbit immunologlobin for the appropriate primary antibody.
Images were collected with a Leica TCS 4D confocal microscope (Leica Microsystems, Bannockburn, IL) equipped with argon, krypton, helium/neon, and UV lasers. Each channel was scanned separately using a 40X oil immersion lens with a numerical aperture of 1.25, and optical sections were saved as digital graphics files. Differential interference contrast (DIC) images of tissue labeled with Quick-Stain were collected with a Nikon Eclipse microscope (Melville, NY) equipped with a Spot CCD camera (Diagnostic Instruments, Sterling Heights, MI). Brightness and contrast were adjusted using Photoshop (Adobe Systems, San Jose, CA)
Paired t-tests were used to determine whether differences between the treated and fellow control eyes (or right and left eyes in the normal group) were statistically significant. There were no significant differences between the normal right and left eyes for any of the four proteoglycans studied. Therefore, an average value for the normal group was obtained by first averaging the right and left eye values for each animal in the group then taking the average of those values for the group. Analyses of variance (ANOVA) and least significant difference (LSD) post hoc tests were used to test whether differences between normal eyes and the treated or control eyes were significant.
Refractive and anatomical changes
The refractive and axial changes produced by minus lens wear and recovery are shown in Figure 1. As expected, four days of monocular-5 D lens treatment produced a myopic shift in refractive state (-2.2±0.5 D, paired t-test, p<0.05) and an increase in vitreous chamber depth (0.07±0.02 mm, mean±SEM, paired t-test, p<0.05) in the treated eyes relative to the control eyes. After two days of recovery, the difference in refractive state had decreased significantly from -4.9±0.2 D at the time of goggle removal to -4.1±0.2 D (paired t-test, p<0.05) indicating that refractive recovery had begun. A-scan measures were not performed in the recovery animals at the end of 11 days of -5 D lens treatment to insure that the general anesthesia required to perform the A-scan measures would not affect the subsequent recovery. There were no significant differences in refractive state or ocular component dimensions between the right and left eyes in the normal group (paired t-test, p>0.05). Additionally, the control eye values were not significantly different from average age-matched normal values.
Figure 2 displays the mRNA levels (mean±SEM) in the treated, control, and normal eyes as copies per million copies of 18s. There was not a significant difference between the right and left eye mRNA levels for any of the four proteoglycans in the normal animals (paired t-test, p>0.05).
Aggrecan mRNA levels showed both bidirectional differential regulation and binocular regulation (Figure 2). After four days of -5 D lens wear, the levels in the treated and control eyes were both significantly lower than the level in the normal eyes (treated eye, -60%; control eye, -43%; ANOVA, LSD, p<0.05). In addition, the level in the treated eyes was 30.8%±2.4% lower than in their fellow control eyes after four days of minus lens wear and 51.4%±2.4% higher after two days of recovery (paired t-test, p>0.05). That the level in the treated eyes was 60% lower, compared to normal eyes, after four days of -5 D and 47% higher than normal after recovery (ANOVA, LSD, p<0.05) suggests that there was absolute bidirectional regulation in the treated eye and not simply relative regulation that could have been due to a change in the control eye. It is also noteworthy that the level in the treated eyes was approximately four-fold higher after two days recovery than after four days of -5 D lens treatment.
As shown in Figure 2, decorin showed the least regulation of the four proteoglycans studied. After four days of -5 D lens treatment, decorin mRNA levels were not significantly different in the treated versus control eyes nor did these eyes differ significantly from normal eyes. The only statistically significant effect was that after two days of recovery, mRNA levels were lower in the treated eyes relative to the control eyes (-13.7%±4.4%; paired t-test, p<0.05). However, at this time point, neither the treated or control eye levels were different from normal eye levels.
Biglycan and lumican mRNAs showed binocular regulation (Figure 2) but little differential regulation. Biglycan and lumican mRNA levels were lower in both the treated and control eyes after four days of lens treatment compared to normal-eye levels (biglycan: treated eye, -55%, control eye, -52%; lumican: treated eye, -52%, control eye, -54%; ANOVA, LSD, p<0.05). After two days of recovery, mRNA levels of both biglycan and lumican were significantly higher than after four days of lens treatment (biglycan: treated eye, 145%, control eye, 130%; lumican: treated eye, 138%, control eye, 173%; ANOVA, LSD, p<0.05). Biglycan mRNA levels showed a small differential effect after four days of -5 D lens treatment; the level in the treated eye was 6.1%±1.4% lower than in the control eye (paired t-test, p<0.05).
Immunohistochemical labeling of tree shrew sclera with antibodies directed against type I collagen and against aggrecan is shown in Figure 3. Type I collagen reactivity varies in intensity, possibly due to variable penetration of the collagen antibody into the interior of the lamellae or partial separation of the lamellae, but is essentially continuous throughout the sclera (Figure 3E,H). In contrast, the aggrecan reactivity is much less continuous and appears to be largely restricted to relatively thin bands (Figure 3F,I). The overlay images (Figure 3G,J) show that much of the collagen reactivity remains discrete (red) while much of the aggrecan reactivity appears to be colocalized (yellow) with collagen. In locations where a fibroblast process was not present, the lamellae are essentially in direct contact. If there is aggrecan in these locations, it would likely appear to be colocalized with the collagen at the surface of the lamellae. This pattern of colocalization suggests that aggrecan is in close proximity to collagen but primarily present in relatively thin bands and not distributed throughout the interior of the lamellae, which is consistent with aggrecan being largely localized to the limited space between lamellae. Voids with no collagen or aggrecan labeling (arrows in Figure 3H,I,J) are likely where the thin optical section passes through the interior of fibroblast bodies and processes. This can be seen most clearly in Figure 3J where rings of discrete aggrecan immunoreactivity appear to surround the regions with no immunoreactivity (arrowheads). There was essentially no immunoreactivity on negative controls (inserts in Figure 3C,H,I). Tree shrew knee cartilage, processed in parallel with scleral tissue served as a positive control for aggrecan, showed abundant aggrecan immunoreactivity (Figure 3C). In summary, these data suggest that aggrecan is present in tree shrew sclera and that it is located primarily between the collagen lamellae rather than throughout the collagen lamellae on the surface of all individual collagen fibrils like decorin .
Several factors suggest that the anti-aggrecan antibody used in this study was specific for aggrecan. First, aggrecan is known to be present in human sclera , and the presence of aggrecan mRNA strongly suggests that aggrecan is present in the tree shrew sclera. Second, the polyclonal antibody used was made to recombinant human aggrecan, amino acids 20-675, which comprises the G1, IGD, and G2 domains. Versican, a proteoglycan that shows some homology to aggrecan and may also be present does not have the G2 domain. The partial known amino acid sequence for tree shrew aggrecan (amino acids 351 - 492, AAH36445.1) which is within the IGD domain shows 81% homology to human aggrecan and no homology to versican. A previous study that used this antibody found that immunoblots of protein obtained from a chondrocyitic cell line showed a single aggrecan-immunoreactive band, indicating that this antibody is specific to human aggrecan . Finally, the aggrecan antibody showed strong reactivity in tree shrew knee cartilage where aggrecan is certainly present. Taken together, these factors suggest that when used, the antibody accurately showed the distribution of aggrecan in tree shrew sclera.
The data of the present study support those of previous studies, which found scleral proteoglycan synthesis was downregulated in tree shrew sclera during experimentally induced myopia and upregulated during recovery [16,23]. In addition, this study provides data on the regulation at the mRNA level of the core proteins of individual proteoglycans. In general, we did not find evidence of strong differential regulation of the three SLRPs that were examined. Decorin, which is abundant in fibrous connective tissue and thought to play a role in controlling fibril diameter and organization , showed neither differential nor binocular changes after four days of -5 D lens wear and only a small differential downregulation after two days of recovery. A previous study that examined scleral decorin mRNA levels during form deprivation-induced myopia and recovery also found little evidence of decorin modulation . These mRNA data are consistent with the finding that dermatan sulfate GAGs, associated with decorin, do not change as a function of scleral dry weight during minus lens wear or recovery . Due to its abundance in fibrous connective tissue and the well-established small decrease in collagen abundance during minus lens wear , we initially hypothesized that changes in decorin content might contribute to the changes in creep rate. However, we have now found that neither form deprivation nor minus lens treatment induces a significant change in decorin mRNA levels suggesting that the observed changes in creep rate are not due to a differential change in decorin content.
Lumican showed binocular changes but no differential ones that might contribute to the differential changes in the mechanical properties of the sclera. However, the small differential changes found in mRNA levels for biglycan could potentially contribute. Overall, the three small proteoglycans studied did not show strong differential effects that might be expected if they were to contribute to the strong differential effects on the creep rate of the sclera that occur during minus lens wear and recovery.
In contrast, aggrecan, which is generally considered to be a cartilage proteoglycan, is not only present in tree shrew sclera but also showed strong differential, bidirectional modulation of its mRNA in response to minus lens treatment and recovery: downregulation during myopia development and upregulation during recovery. Regulation of aggrecan has been found previously in the cartilaginous layer of the chick sclera during induced myopia . However, the change is opposite in direction to that found here in the fibrous tree shrew sclera. The opposite effect on aggrecan synthesis in the cartilaginous chick sclera and the fibrous tree shrew sclera is consistent with the finding that the chick cartilaginous sclera grows during myopia development while tissue remodeling alters the mechanical properties of the sclera in the tree shrew [16,45]. It is intriguing that aggrecan synthesis is apparently modulated in opposite directions in the avian and mammalian sclera in response to the same visual stimuli to produce the same affect on axial elongation. It is not yet known whether the same signaling processes have the opposite effect on chondrocytes (raising aggrecan production during myopia development) and fibroblasts (decreasing aggrecan mRNA during myopia development) or whether different signaling molecules are involved in chicks and tree shrews.
Binocular changes such as those seen for biglycan, lumican, and aggrecan have been observed previously for other mRNA levels after monocular minus lens treatment in tree shrews . The mechanism that produces mRNA level changes in the untreated control eye is unknown. While mRNA changes that occur in both the treated and control eye may contribute to the overall effect in the treated eye, it is clear in this study that mRNA changes in the control eye were not sufficient to cause a change in axial and refractive properties within the level of our ability to resolve them. While the differential changes in aggrecan suggest it may be a key component in the modulation of scleral creep rate, a change in aggrecan content alone or in concert with changes in biglycan and lumican (all three were downregulated in the control eye compared to normal eyes during -5 D lens treatment) was not sufficient to cause a change in axial length and refractive state. This suggests that there are other critical components such as the observed changes in HA and collagen that must also occur to produce the axial and refractive changes that occurred in the treated eyes.
Aggrecan levels and scleral mechanical properties
Relatively little is known about the location and function of aggrecan in fibrous connective tissue such as skin and sclera likely because it has not generally been considered to be an important structural element, compared to collagen. In contrast, aggrecan has been studied extensively in cartilage where it plays a critical role in the ability of cartilage to withstand compressive forces . Aggrecan has been found in fibrous tendon but only in regions that undergo compression . Previous immunoelectron microscopic results in human sclera  suggest that aggrecan is localized between collagen fibrils and in the space between adjacent collagen lamellae while decorin and biglycan are located throughout the lamellae in close association with the surface of the collagen fibrils. The aggrecan immunohistochemical data presented in this study generally support that finding. Aggrecan appears to be located primarily around the fibroblasts and between the lamellae and not within the lamellae. The apparent concentration of aggrecan between the lamellae suggests that a reduction of the amount of aggrecan during myopia development and an increase during recovery could contribute to the observed changes in scleral creep rate by facilitating or retarding the lateral slippage of lamellae in response to constant tensile force. It also is of interest that hyaluronan, which is often closely associated with aggrecan, shows a rapid decrease during myopia development and rapid return to normal in recovery in tree shrews . Further, integrin mRNA levels in tree shrew sclera are altered during experimentally induced myopia which may alter fibroblast communication with collagen and other ECM components located between the lamellae .
In addition to modulation of scleral aggrecan mRNA levels, modulation of aggrecan degradation may also be involved in controlling aggrecan content in the sclera during minus lens induced myopia development and recovery. In a previous study , we showed that MT1-MMP and TIMP-3 mRNA levels were differentially regulated during minus lens treatment and recovery in a manner that is consistent with an increase in MT1-MMP activity during lens compensation and a subsequent decrease during recovery. MT1-MMP is a membrane bound MMP that is known to degrade aggrecan , and its location on the fibroblast surface places it between the collagen lamellae where aggrecan appears to be concentrated. The simultaneous modulation of aggrecan synthesis and degradation could produce rapid changes in aggrecan content in the inter-lamellar space that could be a factor in the rapid changes in creep rate. If so, this would be a novel functional role for aggrecan in fibrous connective tissue that is of general interest to tissue biomechanics beyond the field of myopia research. Together, these data on the location, synthesis (at the mRNA level), and possible degradation of aggrecan suggest that key changes involved in the regulation of creep rate may occur at the interfaces of the lamellae rather than within the lamellae.
In conclusion, this study suggests that there is selective modulation of the synthesis of some but not all proteoglycans during experimentally induced myopia and recovery. In concert with previously described changes in type I collagen expression and content [12,16] and hyaluronan content , the modulation of aggrecan, which appears to be located primarily between the collagen lamellae, may play a key functional role in the rapid modulation of scleral creep rate, which in turn may modulate axial elongation rate and refractive state.
Supported by National Eye Institute Grants RO1 EY05922, RO1 EY07845 and P30 EY03039 (CORE). The authors have no commercial interest in the subject matter of the manuscript. We thank Mr. Joel Robertson for assistance in the preparation of the animals used in this study and Drs. Thomas T. Norton and Kent T. Keyser for advice and mentorship. Data in this paper were presented at the Association for Research in Vision and Ophthalmology (2005) E-abstract 3335.
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