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Figure 9. Alterations in TGF-b isoform gene expression during myopia development. Monocular deprivation of form vision was used to induce myopia in tree shrews and TGF-b isoform expression was quantified after one (A, C) and five (B, D) days deprivation. Copies of individual isoforms were quantified in scleral samples (n = 6) with reference to an external standard, and were expressed per 1000 copies of the housekeeping gene, HPRT (A, B). Data is also presented as the percentage difference in gene expression (treated eye – control eye) ± SEM (C, D).* indicates a statistically significant result. (Reproduced with permission from Jobling et al., 2004 © The American Society for Biochemistry and Molecular Biology, Inc.)

of the TGF-B isoforms in the sclera are differentially reduced in an isoformand time-dependent manner possibly reflecting isoform-specific roles in the remodelling of the scleral ECM at different stages of myopia development (see Fig. 9).

Regulators of scleral myofibroblast differentiation

Fibroblast to myofibroblast differentiation is a complex process, with a number of signalling factors important in the fibroblast moving through the proto-myofibroblast to mature myofibroblast stage.58 However, at a basic level the process is initiated either by induced stress on the cell and matrix, or through stimulation with cell signalling factors, among the

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most important of which is the cytokine transforming growth factor beta (TGF-β).58 The sclera, itself, is under constant and fluctuating stress due to intraocular pressure, while TGF-β is present within the scleral matrix and has been implicated in the remodelling that occurs during myopia development. In vitro cell culture studies using attached or stressed collagen gels have shown that scleral myofibroblasts are readily formed by increasing matrix stress. Similarly, the addition of TGF-β to scleral cell cultures brings about a rapid differentiation of fibroblasts into α-SMA- expressing myofibroblasts (Fig. 10).37 Careful assessment of the structural proteins within the cell cytoplasm shows that ‘stress fibers’ have developed within the cell that typically orient themselves parallel to the imposed stress.37

Myofibroblast-extracellular matrix interactions

Myofibroblasts are capable of modifying their extracellular environment both through contraction and the production of new extracellular matrix. Once formed, myofibroblasts produce collagen, proteoglycans and many other constituents and regulators of the extracellular matrix, in order to maintain or repair their extracellular environment. For this reason, myofibroblasts must be continually receiving information on the surrounding matrix. The major significance of this direct cell-matrix interaction is twofold. Firstly, the cell is in a position to immediately sense any changes in the stress experienced by the extracellular matrix, and thus be in a position to change its production and regulation of the extracellular matrix accordingly. Secondly, the cell is in a position to physically respond to any imposed stresses, via contraction of its surrounding matrix.

Data from many different tissue systems show that extracellular matrixproducing cells, such as myofibroblasts, are closely related to their matrix through a variety of cell-matrix adhesion molecules. On the outside of the cell these adhesion molecules act as receptors, binding to various aspects of the extracellular matrix, such as collagen.59 These cell adhesion molecules also span the cell membrane and join, internally, to the cytoskeleton of the cell, forming a complete bridge between the extracellular matrix and the actin of the internal framework of the cell.59 The integrin family of receptors are perhaps the most important cell adhesion molecules in extracellular matrices such as the sclera. Collagen-binding integrins have been demonstrated on scleral cells.31 Of further interest, integrin gene expression has been shown to be altered in eyes developing myopia,

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Figure 10. TGF-β regulation of scleral fibroblast differentiation. Cultured scleral fibroblasts were incubated with (C) or without (A) TGF-β for five days. The expression of the myofi- broblast-marker, α-SMA was assessed using fluorescent immunocytochemistry (×400). Cells observed at higher magnification (×630; E) show α-SMA-containing stress fibers. The respective negative controls are included in panels B and D, and bars represent 50 µm. (Reproduced with permission from McBrien et al., 2009 © American Academy of Optometry.)

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suggesting that the cell-matrix bond is altered in myopic eyes.31 Such a reduction in cell-matrix contact would have implications for the biomechanical response of the sclera.

Cellular and matrix contributions to altered scleral biomechanics and myopia

From the above discussion, scleral myofibroblasts must be considered an integral part of the biochemical and biomechanical response of the sclera, both in normal and abnormal eye growth. These cells certainly contribute to the matrix changes widely reported in the sclera of eyes developing myopia,19 and their mechanical interaction with the matrix, together with their contractile capability, indicate a mechanism whereby the sclera may control its elastic response to short term changes in stress, such as during fluctuations in intraocular pressure due to cardiac cycle, respiration, and eye movement.

A proposed model for the role of scleral myofibroblasts in myopic eye growth, incorporating the current data, is shown in Fig. 11. A retinoscleral signalling mechanism43 initiates a process of scleral tissue loss, partly due to reduced synthesis of extracellular matrix components and partly a result of accelerated degradation.60 As the sclera thins, a series of gene expression changes are initiated amongst the scleral myofibroblasts, which results in the changes in the collagenous matrix that subsequently manifest in myopia development, such as reduced diameter of collagen fibrils.60 Changes in scleral thickness and the material properties of the sclera increase the capacity of the sclera to creep under normal intraocular pressure, and this process also increases the stresses present within the matrix, and therefore on the myofibroblasts. Downregulation of integrin expression early on in the process of myopia development57 represents a mechanism whereby myofibroblasts disconnect from the scleral matrix, releasing the cells’ mechanical influence on the matrix and enhancing the capacity of the sclera to creep and the eye to grow. Such a response may also reflect a protective mechanism in response to the stresses the fibroblast is experiencing. These scleral myofibroblasts may try to reconnect with the creeping matrix, perhaps enhancing their contractile capabilities in doing so. Similarly, they may remain disconnected from the matrix, de-differentiating to fibroblasts, due to their reduced experience of the stress in the matrix, and allowing further increase in the creep capacity of the sclera (Fig. 11).

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Figure 11. Proposed schematic model of the role of scleral myofibroblast cells in the biochemical and biomechanical remodelling that facilitates the scleral changes that occur during myopia development and progression, based on current evidence. See text for details. (Reproduced with permission from McBrien et al., 2009 © American Academy of Optometry.)

The biomechanical properties of the sclera are critical in maintaining normal ocular development. Alterations in these properties, such as those seen during myopia development, produce concurrent alterations in eye size. While remodelling to the scleral matrix was considered to be the sole determinant of biomechanical change, recent data has highlighted the important role of scleral cells, particularly scleral myofibroblasts. While our current knowledge of the role of scleral myofibroblasts in normal and abnormal eye growth is incomplete, proper identification of the factors involved in scleral weakening and subsequent increased eye size