Abstract
purpose. The sclera has a collagen-rich extracellular matrix that undergoes significant biochemical and biomechanical remodeling during myopic eye growth. The integrin family of cell surface receptors play critical roles in extracellular matrix and biomechanical remodeling in connective tissues. This study identified the major collagen-binding integrin receptors in the mammalian sclera and investigated their mRNA expression during the development of and recovery from experimental myopia.
methods. The presence of the α1, α2, and β1 integrin subunits was examined by using tree-shrew–specific primers and RT-PCR. Scleral expression of α1β1 and α2β1 receptor proteins was further investigated by using Western blot analysis and immunocytochemistry. Myopia was induced monocularly by occluding pattern vision and scleral tissue collected after 24 hours and 5 days. In a subset of the 5-day treatment group, vision was restored for 24 hours before tissue was isolated. Total RNA was extracted, and integrin subunit expression levels were assessed with quantitative real-time PCR.
results. The presence of the major collagen-binding integrin subunits α1, α2, and β1 was confirmed by RT-PCR in both scleral tissue and cultured scleral fibroblasts. Both the α1 and α2 integrin subunit proteins were identified in tree shrew scleral tissues, and integrin receptor expression was localized to scleral fibroblast focal adhesions. After only 24 hours of myopia induction, a time when no structural elongation has occurred, significant decreases were observed in the expression of the α1 (−36%) and β1 (−44%) integrin subunits. After 5 days of myopia induction, α1 integrin expression had returned to baseline levels, whereas the α2 subunit showed a significant decrease in expression (−52%). The 5-day integrin profiles were maintained during recovery from the induced myopia, with only α2 integrin showing a statistically significant relative decrease in expression (−41%).
conclusions. The mammalian sclera expresses the major collagen-binding integrin subunits. The α1 and β1 subunit expression was decreased early during the development of myopia, whereas the regulation of α2 integrin occurred at a later time point. The differential regulation of α1β1 and α2β1 during the development of myopia may reflect specific roles for these receptors in the scleral extracellular matrix and biomechanical remodeling that accompanies myopic eye growth.
High myopia is characterized by excessive elongation of the eye, particularly in the axial dimension, and results in an increased risk of retinal and/or choroidal disease.
1 During the development of myopia the outer coat of the eye, the sclera, undergoes an active remodeling process, which results in a progressive thinning and weakening of the tissue. It appears to be this remodeling of the scleral extracellular matrix (ECM)
1 that facilitates the abnormal increase in eye size.
The scleral ECM is predominantly composed of type I collagen, contains several different proteoglycans, and is maintained by a population of fibroblast cells.
2 The remodeling that occurs as myopia develops, is characterized by changes in collagen synthesis, collagen degradation, and fibril diameter.
3 4 5 In addition, alterations have been observed in matrix metalloproteinase, glycoprotein, and growth factor levels (Norton TT et al.
IOVS 1995;36:ARVO Abstract 3517)
6 7 Although it is still not fully substantiated what initiates and regulates the scleral remodeling, studies in other fibroblast-maintained tissues undergoing ECM remodeling have highlighted the importance of cell-ECM signaling.
8 The integrin family of cell surface receptors are known to play a critical role in such communication.
Integrins are heterodimeric, transmembrane receptors, formed from the noncovalent association of an α and β subunit. Presently, there are 18 α and 8 β subunits that are known to form 24 distinct integrin receptors.
9 Each receptor binds a specific ECM ligand, although different integrin receptors can bind the same ligand.
10 This apparent redundancy is not reflected in vivo, indicating a further level of complexity beyond the receptor subunit composition.
11
Integrins have wide-ranging cellular effects. Initially discovered because of their role in linking the cell to the surrounding ECM,
10 12 integrins have subsequently been shown to regulate diverse cellular functions such as survival, proliferation, migration, and differentiation.
13 14 15 16 Their role in cellular adhesion also involves integrin receptors in mechanotransduction, the process by which cells convert mechanical forces into biochemical signals.
17 18 Such regulation is particularly important for those tissues that are under constant mechanical load and enables cells to respond to changes in the ECM stresses.
19 Integrins also share a close relationship with several growth factor receptors, such as the epidermal growth factor receptor (EGFR) and those of platelet-derived growth factor (PDGFR) and vascular endothelial growth factor (VEGFR-2),
20 21 22 with data even showing that growth factor signaling can be activated by integrins, independent of growth factor ligand.
23
Because of the important and wide-ranging effects of integrins, it is not surprising to find that these receptors are expressed within the eye. Several ocular tissues such as cornea, lens, retina, and choroid have been shown to express integrins.
24 25 26 By far the best characterized is the cornea, where integrins have roles in the maintenance of corneal integrity and epithelial wound healing.
24 27 In addition, these receptors have been implicated in neovascular retinal disease and retinal development.
28 29
The role of integrins in ocular growth, however, is less evident. As the sclera has a high ECM content, is under constant tension due to intraocular pressure and is regulated by growth factors, integrins are likely to be involved in the regulation of this tissue during eye growth. There are very limited data on integrin expression in the sclera, with only one study reporting immunohistochemical staining for the β1 subunit in
Xenopus scleral fibroblasts.
30 Recently, we reported the presence of 13 integrin receptors in the mammalian sclera, including the collagen-binding receptors, α1β1, α2β1, α10β1, and α11β1.
31 While there are limited data on the functional roles of the α10 and α11 collagen receptors, the α1β1 and α2β1 receptors are considered the major collagen-binding integrins and have been extensively studied.
32 33 The expression of these receptors in the sclera are of particular interest, because numerous reports have detailed alterations in collagen synthesis, degradation, and structure during the ocular elongation that results in myopia.
4 5 34
The present study addresses the lack of data on integrin expression within the mammalian sclera by identifying the major collagen-binding integrins α1β1 and α2β1 and characterizing changes in their expression during abnormal eye growth. As the sclera has a collagen-rich ECM and significant alterations in collagen turnover have been reported in animal models of myopia, alterations in these specific integrin receptors are highly likely to play a critical role in the scleral remodeling that accompanies myopia.
After the specific treatment periods, animals were anesthetized (ketamine 90 mg/kg, xylazine 10 mg/kg), and a lethal dose of pentobarbital sodium (120 mg/kg) was administered before tissue collection. Left eyes were enucleated first, to randomize the processing of the treated eye. An incision was made posterior to the limbus, and the anterior segment containing the cornea and lens was removed. The eye cup was flatmounted, and the retina and choroid were dissected. For gene expression studies, a 7-mm scleral punch was isolated by using a surgical trephine and the optic nerve head was removed. Scleral tissue was immediately placed in liquid nitrogen and subsequently stored at −80°C.
For primary scleral fibroblast cultures, whole sclera was isolated and placed in a culture vessel (Nunc, Roskilde, Denmark) containing Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS), 25 mM HEPES, and 100 U/mL penicillin and streptomycin (JRH, Melbourne, Australia). Cell outgrowth from explants was observed after ∼1 week, and confluence was generally reached after 3 weeks. Cultures were passaged using 0.25% trypsin (Invitrogen), and cells between passages 2 and 5 were used. Primary skin fibroblast cultures were used as a positive control for integrin subunit expression and were established from a lateral skin flap that had been cleaned of excess fat and minced. Growth medium was identical with that described earlier.