Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

dependent on vision.104 Fish (Tilapia), which are more vision-dependent, display this modulation characteristic.105 Interestingly, for negative lens compensation to occur in animals, the lens must be worn almost constantly. Removing the negative lens and allowing normal unrestricted vision (or plano-lens wear) for as little as 2 hours per day is sufficient to block negative lens compensation in monkeys,106

tree shrews,107 and chicks.108

Plus lenses

Plus lenses shift the focal plane anteriorly, converting an emmetropic eye into one that is myopic. In examining the effect of plus lenses in animal studies, it is important to note that they are applied to juvenile eyes that are still undergoing normal axial elongation. If the axial elongation rate is slowed below normal while the optical components continue to mature normally, the eye gradually becomes emmetropic while wearing the plus lens. With the lens removed it is hyperopic. Thus, to respond to a plus lens, the eye must not only be able to detect that it is myopic, but also be able to slow its elongation rate. In a young, growing chick eye, plus lens wear quickly causes a decrease of the elongation rate such that the eye becomes emmetropic while wearing the lens and hyperopic when the lens is removed.96,98 Even short exposures to myopic defocus have a potent slowing effect.108–112

The response to plus lenses differs somewhat in monkeys and tree shrews. When plus lenses are applied to tree shrews that have achieved emmetropia, little effect is noted.113 However, if plus lenses are applied early in the emmetropization process in younger, hyperopic tree shrews and monkeys, the elongation rate of the eyes decreases, and emmetropia is maintained while wearing the lens.99,114–116 With lens removal, the eye remains hyperopic. The lack of a response to plus lenses in older tree shrews and monkeys may be due to an inability to slow axial elongation rate substantially below normal once the eyes have achieved emmetropia. The source of this difference in the chick is unknown, but may be related to different sclera composition in the avian eye relative to that of the mammalian. The chick eye sclera has an inner cartilaginous layer in addition to the fibrous sclera layer present in humans, monkeys, and tree shrews. Controlling the growth of the cartilage may be a more powerful way of controlling axial elongation than can be achieved with a fibrous sclera alone.

Recovery from induced myopia

Further evidence for active control of axial length growth comes from the effect of removing form deprivation or negative lens wear after an induced myopia has developed. In the several species that have been studied, “recovery” from induced myopia occurs. Restoration of unrestricted vision causes the axial elongation rate to decrease below normal. As the focal plane moves posteriorly due to continued corneal flattening and lens maturation the induced myopia dissipates.67,103,117–119 It has been suggested that recovery occurs for two reasons: first, the eye is myopic, and second, it is elongated relative to normal. These factors together produce a stronger response than is produced by plus lens wear alone.120 In animals with monocular induced myopia, the refractive state of the recovering eye eventually matches that of the untreated fellow control eye at emmetropia, suggesting that active guidance occurs moving towards a “target” of emmetropia. The emmetropization mechanism is thus capable of acting to control axial elongation to achieve emmetropia from the myopic, as well as the hyperopic, direction. The consistency of responses to form deprivation, negative lens wear, and recovery provides evidence that an active emmetropization mechanism exists and is conserved across phyla. Together with the rapid development in human infants of a very narrow refractive error distribution, these data argue strongly that an emmetropization mechanism exists in humans.

The emmetropization feedback loop

The signaling cascade

How does the retina control the axial elongation rate to match the retina to the focal plane and maintain that match throughout the juvenile developmental period? The broad outline of a feedback loop has emerged from studies of animal models. The starting place is the visual stimulus (1 in Figure 55.3). Although it is not precisely known what aspect of the images on the retina stimulates elongation, there is general agreement that hyperopic defocus (image plane behind the retina) serves as a stimulus for eyes to increase the axial elongation rate (“go” signal). Myopic defocus, or perhaps clear images projected on the retina,

Figure 55.4  Structure of the mammalian sclera. Model illustrating the likely biochemical structure of the extracellular matrix of the mammalian sclera. FGF-2, fibroblast growth factor-2; TGF-β1, transforming growth factor-β1; MMP, matrix metalloproteinase; TIMP, tissue inhibitors of metalloprotease. (Adapted from Fransson et al.157)

produces signals that slow axial elongation (“stop” signal). Defocus, or its absence, is detected by the retina (2 in Figure 55.4), presumably at the level of center-surround bipolar cells or later.121,122 As has been established by single-unit studies of retinal ganglion cells,123 defocused images produce weaker responses from cells with center-surround receptive fields than do sharply focused images. Thus, as the eyes move from target to target, with varying retinal image stimulation, focused images will produce large changes in the retinal cell activity level.

In chicks, a class of amacrine cells that contain glucagon are involved in sending a “stop” signal to slow the eye elongation rate of the eye.124–126 Without these cells, eyes elongate and become myopic. With them, and with additional glucagon administered intravitreally,126 eyes resist becoming myopic with form deprivation. It has also been reported that the protein apolipoprotein A1 provides a retinal “stop” signal in chicks.127

Perhaps surprisingly, the emmetropization mechanism persists after sectioning of the optic nerve,128 or blocking the output from the retina to central visual structures with tetrodotoxin,129,130 leading to the conclusion that there is direct communication across the RPE and choroid (3 and 4 in Figure 55.3) to the sclera of the eye. The communication to the sclera must also be spatially local. When form deprivation or negative lens treatment is applied to half of the visual field (either nasal or temporal) using specially designed goggles, only the treated half of the eye elongates and becomes myopic.131–134 More surprisingly, the fovea is neither necessary nor sufficient to establish and maintain emmetropia. Macaque monkeys that underwent laser destruction of the entire macular region maintain emmetropia.135 Monkeys that wear form-depriving diffuser goggles with a clear central 30° to allow foveal vision develop form deprivation myopia.135,136

How the visually derived signals pass through the RPE is unknown. It has been suggested137 that changes in retinal activity produce changes in ionic concentrations across the RPE that in turn affect the tissues of the choroid. This part of the emmetropization signaling cascade is under active investigation. Mertz and Wallman138 suggested that

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all-trans-retinoic acid in the choroid may serve as a signaling molecule to the sclera. Rada et al139 have suggested that ovotransferrin might also serve a similar role.

Visual response remodeling of the sclera

As the structural outer coat of the eye, the sclera controls the location of the retina. The sclera in eutherian mammals is a soft, but tough, fibrous viscoelastic connective tissue comprised of an extracellular matrix (ECM) produced by fibro­ blasts that are of neural crest origin.140–142 It consists of layers, or lamellae, composed of collagen (85–90% of the scleral total protein), associated proteoglycans, hyaluronan, and

other extracellular proteins produced by the fibroblasts. Type I collagen is the primary subtype (>99%)143 but types III, V,

and VI are present in tree shrew sclera.115,144 Unlike the corneal stroma, the diameter and spacing of the collagen fibrils vary widely, so that the sclera is not transparent. In addition to collagen, elastic fibrils comprised of elastin and laminin have been reported in the human sclera.145 The proteoglycans aggrecan, biglycan, and decorin are also present in the adult human sclera146 and in tree shrew sclera.147 Austin et al148 suggested that another proteoglycan, lumican, is important in the formation of scleral collagen fibrils.

Glycosaminoglycans (GAGs) constitute a small (~0.2%), but potentially important, fraction of the scleral dry weight. Functionally, GAGs have been suggested to stabilize the spacing of collagen fibrils149 and to affect the hydration levels

of ECM.150,151

In addition to these structural components, the sclera contains several growth factors (e.g., transforming growth factor-ß, fibroblast growth factor-2)152 as well as degradative enzymes (matrix metalloproteinases and tissue inhibitors of metalloproteinases).153,154 A simplified scleral structure is provided in Figure 55.4.

After completion of the infantile growth period, there is little evidence in mammals for continued scleral remodeling.143 Rather than modulating cartilage growth (as occurs in chicks155,156), the visual environment controls remodeling of the scleral ECM. This discussion will focus on mammalian sclera, with particular emphasis on tree shrew because it has been the subject of considerable research.

When “go” signals reach the sclera, changes in expression for some genes occur (5 in Figure 55.3), along with remodeling (6 in Figure 55.3) of the scleral ECM.158 In tree shrews, and perhaps in mammals generally, these biochemical changes appear to control the biomechanical property of sclera, creep rate (6 in Figure 55.3). The creep rate is the extension of the sclera over time when a constant tension is applied. Normally in juvenile tree shrew eyes, scleral extensibility is low. As a result of the remodeling of the sclera, the creep rate rises.100 Loosely speaking, the sclera becomes more readily extensible. The changes in creep rate in turn allow normal intraocular pressure to increase the axial elongation rate of the eye (8 in Figure 55.3), moving the retina away from the cornea. In a hyperopic eye an increased axial elongation rate reduces the hyperopia, decreasing the defocus on the retina. Through this emmetropization feedback system, a hyperopic eye elongates until the visual signals (1 in Figure 55.3) that drive the feedback loop diminish and the eye stabilizes at emmetropia.

Scleral changes during myopia development and recovery

Many of the details of how the visual environment controls scleral remodeling summarized in the model shown in Figure 55.3 emanate from studies using form deprivation or negative lenses to induce the eyes to become myopic. Both interventions (“treatments”) produce a visual signal that stimulates the feedback loop, raising the amount of scleral remodeling, increasing the creep and axial elongation rates. Changes observed during recovery are generally the reverse of those found during myopia development.

When myopia is induced, the sclera becomes thinner, due in part to a reduction in the amount of type I collagen.141,157–159 Reductions of dry scleral weight of 3–5% develop after a few days of form deprivation or negative lens wear in tree shrews.159 Long-term form deprivation in tree shrew leads to a more substantial thinning, similar to that seen in humans with moderate myopia.160 Humans with pathological myopia have a marked scleral thinning, particularly at the posterior pole.161 In addition to scleral thinning, the collagen fibrils often show altered architecture in high human myopia, with a reduction in diameter, loss of longitudinal fibril striations, and a derangement of the growth and organization of the fibrils.162 Following long-term form deprivation in tree shrews, Norton and Rada158 noted a reduction in hydroxyproline (−11.8%) which suggests a loss of collagen. McBrien et al found similar changes: a reduction in median fibril diameter and misshapen collagen fibrils in tree shrews.153

One advantage of animal models, in contrast to examination of human donor eyes, is that it is possible to examine the sclera and other tissues during the time an induced myopia is developing, rather than studying the postmortem endpoint of a longstanding myopic condition. After shortterm form deprivation, although the sclera is thinner, the collagen fibrils appear normal in morphology, diameter, and spacing.153,163 During recovery, there is an increase in collagen synthesis and in mRNA expression level for type I collagen.115 These findings suggest that more subtle remodeling of scleral tissue occurs rather than destruction of structural elements, causing axial elongation and myopia development immediately after the onset of form deprivation or negative lens wear.

Animal model form deprivation study in relation to human myopia

Although animal models have provided information about how environmentally induced myopia can develop, questions have been raised about the applicability of animal studies to human myopia. For instance, form deprivation myopia has been reported to develop in young children with obstructive visual axis conditions such as ptosis or cata- ract.164–167 The myopic shift from form deprivation is inconsistent in humans, however.168

An important similarity between animals and humans was noted by Gwiazda et al,169 who reported that progressing myopes (children with increasing myopia) underaccommodate to near visual targets more than do emmetropic children. The resulting hyperopic defocus resembles the

Human molecular genetics of myopia

hyperopic defocus that is produced by minus lens wear, which causes axial elongation in many animal species. This observation led to the “blur hypothesis,” which suggests that the eyes of children also elongate in response to the hyperopic defocus that occurs when the child underaccommodates to near targets. To the extent that accurate accommodation occurs, the defocus is reduced, which may be why the children with more accurate accommodation remained emmetropic in the Gwiazda et al study.169 The blur hypothesis is attractive as it not only relates animal studies to the human experience, but is also consistent with studies that have associated myopia development with near work.3,11 It contrasts with the suggestion, never supported by direct measurement, that accommodation itself is a cause of myopia170,171 by increasing tension on the sclera, stretching it so the eye elongates.53 The alternative provided by the blur hypothesis is that accommodation is beneficial, because it provides clear images on the retina and thereby removes the visual stimuli for increased axial elongation that can lead to myopia.

Human molecular genetics of myopia

Ocular refractive component genetics

The refractive state is determined by the relative contributions of the optical components, primarily corneal curvature, anterior-chamber depth, and lens thickness – all of which determine the location of the focal plane, and the axial length (primarily the vitreous chamber depth) – which determines whether the retina is located at the focal plane (Box 55.3). Separately, these may be assessed as quantitative traits intimately related to the clinical phenotype of myopia. Multiple reports have examined familial aggregation and heritability of ocular components.172–181

As expected from the previous sections, axial length is the largest contributor to the determination of refractive error. Several studies have reported an inverse relationship of axial length to refraction (the longer the eye, the more myopic the refractive error).174–178 Axial length of a myopic adult population may show a bimodal distribution with a second peak of increased axial length relating to high myopia (< −6 D at 24 mm, > −6 D at 30 mm) when plotted as a distribution curve.54 This suggests that myopia of −6 D or greater represents a deviation from the normal distribution of axial length and is not physiologic.

Estimates of heritability for axial length range from 40% to 94%.172,179–184 A study of three large Sardinian families found modest evidence for linkage on chromosome 2p24 with a likelihood of the odds (LOD) score of 2.64.181 Overall

Box 55.3  Human myopia molecular genetic studies

Molecular genetics studies of human myopia provide a relatively new translational possibility for determining genetic variants associated with myopia. The groundwork from these types of studies may enable the development of new therapies for treatment or prevention, or provide genetic tests for predicting those at greater risk for accompanying ocular morbidities of myopia

axial length includes anterior-chamber depth, and studies have shown that increased anterior-chamber depth has an inverse relationship as well to refractive error.178 The heritability reports for anterior-chamber depth range from 70% to 94%,173,179–181 and the same Sardinian study found modest linkage evidence to chromosome 1p32.2 with a LOD score of 2.32.181

The steeper the corneal curvature, the more likely the resulting refractive error is myopic – eyes with hyperopia are more likely to have flatter corneal curvature readings by keratometry.174,185,186 Heritability estimates for corneal curvature range from 60% to 92%.173,179–181 The Sardinian family study noted modest linkage evidence of corneal curvature to chromosomes 2p25, 3p26, and 7q22 with LOD scores ranging from 2.34 to 2.50.181 Increased lens thickness correlates with increased myopia.178 A diand monozygotic twin study reported 90–93% heritability for lens thickness.179

Role of environment in myopic development

The prevalence of myopia in some populations appears to have increased dramatically from one generation to the next in progressively industrialized settings, or with increased level of educational achievement.45,187–189 Assessing the impact of inheritance on myopic development may be confounded by children adopting parental behavioral traits associated with myopia, such as higher-than-average nearwork activities (i.e., reading).190 Observational studies of this risk factor do not fully explain the excessive familial clustering of myopia, however. A detailed assessment of confounding effects and interactions between hereditary and environmental influences in juvenile-onset myopia has shown that near work describes very little of the variance in refractive error compared to parental myopia.191 Additionally, near work exerted no confounding influence on the association between parent and child myopia, indicating that children do not become myopic by adopting parental reading habits. More importantly, there was no significant interaction between parental myopia and near work; reading was weakly and equally associated with myopia regardless of the number of myopic parents. This indicates that children could inherit myopia as a trait from parents. Of late, it has been proposed and accepted that relatively decreased outdoor activity, rather than near work, has a greater impact on myopic refractive error development.192

Hereditary factors in myopic development

Multiple familial aggregation studies report a positive correlation between parental myopia and myopia in their children, indicating a hereditary factor in myopia suscepti- bility.193–196 Children with a family history of myopia had on average less hyperopia, deeper anterior chambers, and longer vitreous chambers even before becoming myopic. Yap and colleagues noted a prevalence of myopia in 7-year-old children of 7.3% when neither parent was myopic, 26.2% when one parent was myopic, and 45% when both parents were myopic. This implies a strong role for genetics in myopia.182

Multiple familial studies support a high hereditary basis for myopia, especially higher degrees.183,184,197,198 Naiglin and colleagues performed segregation analysis on 32 French

multiplex families with high myopia, and determined an autosomal-dominant (AD) mode of inheritance.199 The ls for myopia (the increase in risk to siblings of a person with a disease compared to the population prevalence) has been estimated to be approximately 4.9–19.8 for sibs for high myopia (−6.00 spherical D or greater), and approximately 1.5–3 for low or common myopia (approximately −1.00 to −3.00 spherical D), suggesting a definite genetic basis for high myopia, and a strong genetic basis for low myopia.200,201 A high degree of familial aggregation of refraction, particularly myopia, was reported in the Beaver Dam Eye Study population after accounting for the effects of age, sex, and education.202 Segregation analysis suggested the involvement of multiple genes, rather than a single major gene effect.

Twin studies provide the most compelling evidence that inheritance plays a significant role in myopia.55,172,179,184,203 Multiple studies note an increased concordance of refractive error as well as refractive components (axial length, corneal curvature, lens power) in monozygotic twins compared to dizygotic twins.55,179,184,204 Sorsby et al noted a correlation coefficient for myopia of 0 for control pairs, 0.5 for dizygotic twins, and almost 1.0 for monozygotic twins in a study of 78 pairs of monozygotic twins and 40 pairs of dizygotic twins.204 Twin studies estimate a notable high heritability value for myopia (the proportion of the total phenotypic variance that is attributed to genetic variance) of between 0.5

and 0.96.172,179,184,203,204

Molecular genetic studies of human high-grade myopia

Several highly penetrant genetic loci for moderate and high myopia have been mapped (reviewed by Young et al).205 These loci are primarily AD or X-linked. They have been mapped in either large, isolated pedigrees, or populations derived from a limited number of founders that have experienced limited migration. Except for the X-linked MYP1 gene, none of the causative mutations has yet been

found.206–209

Much of the current information on human myopia molecular genetics can be drawn from studies of familial high-grade myopia, usually defined as spherical refractive error > – 5.00 D. An X-linked recessive form of myopia, named the Bornholm (Denmark) eye disease (BED), was designated the first myopia locus (MYP1: OMIM 310460) on chromosome Xq28.86,206 Collaborating with BED researchers, we made comparative molecular genetic haplotype and sequence analyses of a large Minnesota family of Danish descent that showed significant linkage of myopia to chromosome Xq27.3-q28. The phenotype of both families appears to be due to a novel cone dysfunction, and not simple myopia.207 The genetic etiology of each family appears to be distinct, as the haplotypes were different.207 A report by Michaelides et al confirms the different X-linked cone dysfunction syndrome with an associated high myopia phenotype as distinct from the BED in four families.208 The first identified myopia gene – CXorf2 at the MYP1 locus – has a distinct sequence mutation in the promoter region, and is a copy number variant.209

Loci identified to date for isolated nonsyndromic highgrade myopia are primarily AD and highly penetrant. The first AD locus for nonsyndromic high-grade myopia was

mapped within a 7.6-cm region on chromosome 18p11.31 (MYP2: OMIM 160700) in seven US families.210 This locus was confirmed in Chinese Hong Kong and Italian Sardinian cohorts.211,212 Using the Hong Kong cohort, investigators identified transforming growth-induced factor-β (TGIF-β) as the implicated gene for MYP1 using limited singlenucleotide polymorphism (SNP) association studies and exonic sequencing.213 Our group fully sequenced TGIF-β in our cohort of original MYP1 families, and found no associations with high myopia affected status.214 An Australian lab also investigated the association of TGIF with refraction and ocular biometric measurements in a Caucasian case-control population, and found no association.215 No association of TGIF to high-grade myopia case-control studies was found in Japanese and Chinese cohorts.216,217

A second locus for AD high myopia mapped to a 30.1 cM region on chromosome 12q21-23 (MYP3: OMIM 603221) in an American family of German–Italian descent also in our laboratory.218 This locus was confirmed in a high-myopia Caucasian British cohort, and in a large German family.219,220 A statistically suggestive third locus for AD high myopia was reported on chromosome 7q36 in a Caucasian French cohort (MYP4: OMIM 608367).221 A fourth AD locus on chromosome 17q21-23 (MYP5: OMIM 608474) was determined in a large multigenerational English–Canadian family in our laboratory.222 We identified a locus for AD high-grade myopia on chromosome 2q37 (MYP12: OMIM 609994) in a large, multigenerational US Caucasian family.223 This locus was replicated in an Australian study of three multigenerational AD moderate myopia families.224 We determined a new locus (MYP17) on chromosome 10q21.2 in a Hutterite (Austrian/German) colony.225 Loci on chromosomes Xq2325226 (MYP13: OMIM 300613) and 4q22-27227 (MYP11: OMIM 609994) were identified by Zhang et al in ethnic Chinese families. Other high-grade myopia loci have been recently mapped to chromosomes 15q12-13, 5p15.33p15.2, and 21q23.3.228–230 The first locus implicated in ocular axial length, which is a major endophenotype for refractive error, was identified on chromosome 5q.231

Juvenile-onset myopia genetics

At least two studies have shown nominal or no linkage of low to moderate myopia to many of the known high myopia loci. Mutti et al genotyped 53 common myopia families (at least one child with more myopia than −0.75D in each meridian) using the highest intrainterval LOD score microsatellite markers for the chromosome 18p and 12q loci and did not establish linkage.232 Ibay et al found no strong evidence of linkage to the chromosomes 18p, 12q, 17q, and 7q in a cohort of 38 Ashkenazi Jewish families with mild/moderate myopia (−1.00 D or more).233 These studies suggest that different genes account for mild/moderate myopia susceptibility or development, or that the gene effects are too small to be detected with the relatively small sample sizes. However, a recent study showed replication of the chromosome 2q37 (MYP12) locus in a cohort of Caucasian Australians with moderate to low myopia.224

Three whole-genome mapping studies have identified several candidate gene intervals for common, juvenile-onset myopia using spherical refractive error data. The results of these studies demonstrate the potential for determining

Mouse models of myopia

molecular genetic factors implicated in myopia at all levels of severity. These studies used microsatellite genotyping technology and a limited cohort sample size, and two used homogenous isolated populations. One study was a genome screen of 44 families of Ashkenazi Jewish descent.234 Individuals with at least −1.00 D of myopic spherical refractive error were classified as affected. Their strongest signal localized to chromosome 22q12 (MYP6: OMIM 608908) (heterozygous linkage of the odds = 3.56; nonparametric linkage = 4.62). Eight additional regions (14q, 4q22-q28, 8q22.2, 10q22, 11q23, 13q22, 14q32, and 17qter) had nominal linkage evidence. The same group replicated the same locus with additional Ashkenazi Jewish families.235 Hammond et al evaluated 221 dizygotic twin pairs with moderate myopia and found significant linkage to four loci, with a maximum LOD score of 6.1 on chromosome 11p13 (MYP7: OMIM 609256).173 Other identified loci mapped to chromosomes 3q26 (MYP8: OMIM 609257: LOD 3.7), 4q12 (MYP9: OMIM 609258: LOD 3.3), and 8p23 (MYP10: OMIM 609259: LOD 4.1). This group found that the PAX6 gene at the chromosome 11p13 locus showed linkage with 5 SNPs, but no association. They suggested that PAX6 (a major eye development gene) may play a role in myopia development, possibly due to genetic variation in an upstream promoter or regulator. This group later reported that neither of the master control genes PAX6 or SOX2 was implicated in common myopia in a large population study cohort.236 A report replicated the chromosome 8p23 myopia locus (MYP10) in an isolated Pennsylvania Amish population of 34 families.237 Other loci identified include chromosomes 1p36, 1q, 7p15, and 3q26.238–242

Gene associations

High myopia also occurs as a feature of several ocular or systemic disease syndromes in which the causative disease gene is known (Table 55.1). Candidate gene association studies have led to the identification of a number of high- myopia-susceptibility genes (Table 55.2). These findings await replication in independent cohorts.

Mouse models of myopia

Factors that regulate rate and duration of eye growth in mice have revealed two loci (Eye1 and Eye2) that may be respon-

Table 55.1  Selected syndromic disorders featuring high myopia

Section 7  Other Chapter 55  Myopia

Table 55.2  High-myopia-susceptibility genes

sible for larger eye size.250,251 Human syntenic homologous regions are chromosomes 6p, 16q13.3, and 19q13 for Eye2, and chromosome 7q for Eye1.

The first knockout mouse model for relative myopia131 was based on form deprivation experiments in chickens, mice, and rhesus macaque monkeys.252 The immediate early gene transcription factor ZENK (also known as Egr-1) is implicated in the feedback mechanisms for visual control of

axial eye growth and myopia development.253–255 ZENK is upregulated in retinal amacrine cells when axial eye growth is inhibited by positive lens wear, and is downregulated when axial growth is enhanced by negative lenses, suggesting that ZENK is linked to an axial eye growth-inhibitory signal. ZENK knockout mice had longer eyes and a relative myopic shift relative to heterozygous and wild-type mice with identical genetic background.253

Conclusions

The field of myopia science continues to expand with newer techniques of both assessing myopic development and determining molecular shifts. This chapter details research findings for the most common refractive error, myopia, describes human eye growth patterns, describes how animal models have advanced our understanding of the visually guided mechanism that matches the eye’s axial length to its optical power, and discusses the genetic basis of human myopia. Understanding the molecular mechanism that controls axial length in humans is an important step toward developing customized treatments to control axial elongation.

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Clinical background

Graves’ disease (GD) was named for the Irish physician, Sir Robert James Graves (1797–1853), who first described the triad of hyperthyroidism, goiter, and exophthalmos.1 Graves’ hyperthyroidism is caused by targeting and stimulation of the G protein-coupled thyroid-stimulating hormone receptor (TSHR) by TSH receptor autoantibodies, leading to the overproduction of thyroid hormones (Box 56.1).2 After the cloning of the TSHR in 1989, insight regarding pathogenesis of GD grew markedly.3–5 However, while recent strides have been made, the pathophysiology of Graves’ ophthalmopathy (GO) remains less well understood.2

GD has an annual incidence in women of one per 1000 population; the annual incidence of GO in women is 16 in 100 000 and in men 3 in 100 000.6 Approximately 25–50% of patients with Graves’ hyperthyroidism have clinical eye involvement.7 A temporal relationship exists between the onset of Graves’ hyperthyroidism and the onset of GO. In 80% of affected patients, regardless of which condition occurs first, the other condition develops within 18 months.8

GO patients characteristically display symptoms of a dry, gritty sensation in their eyes, blurry vision, photophobia, excessive tearing, diplopia, or a pressure sensation behind the eyes.9 The clinical signs of GO include ophthalmoplegia, lid lag, proptosis, chemosis, corneal ulceration, and conjunctival erythema (Figure 56.1). Computed tomographic scans of the orbits in 90% of GD patients reveal characteristic orbital modifications indicating eye involvement, whether or not clinical signs are present.10 While the majority of GO patients have only mild congestive ocular symptomatology, in 3–5% severe eye disease is present, and compressive ischemic optic neuropathy with vision loss develops in the rare patient.11

Although the majority of GO patients have an increase in both the orbital fat and extraocular muscle volumes, some exhibit primary involvement of only one of these tissue compartments.12 The age of the patient appears to influence the tissues involved as increased orbital fat is more commonly seen in patients younger than 40 years, and enlarged extraocular muscles predominate in patients over 70 years.13 In early disease stages, enlargement of the extraocular muscles is caused by edema and excessive accumulation of

Pathogenesis of Graves’

ophthalmopathy

A Reagan Schiefer and Rebecca S Bahn

hyaluronic acid, leading to intermittent or inconstant diplopia. In later stages of the disease, the muscles may atrophy and become fibrotic due to the chronic inflammatory process, with resultant ocular malalignment and restrictive, constant diplopia.6

Pathology

Histologic examination of orbital adipose and extraocular muscle tissues in GO shows an overabundance of complex carbohydrates termed glycosaminoglycans (GAGs), in which hyaluronic acid predominates. Orbital fibroblasts are the major source of these molecules and these cells are thought to be the autoimmune target in GO.14 In early stages of GO, muscle fibers are grossly intact, but are widely separated by edematous perimysial connective tissues resulting from accumulation of hydrophilic GAGs.15 Similarly, expansion of the orbital adipose tissue compartment is in part attributed to accumulation of GAGs. In addition, the expansion of orbital fat results from the differentiation of a population of preadipocytes within the orbit into mature lipid-laden adipocytes.2

In addition to the excess of GAGs, histologic examination of orbital tissues in GO shows diffuse infiltration of lymphocytes. While these cells are predominantly T lymphocyte, occasional B cells are present as well.2 The existence of activated lymphocytes and cytokines in GO orbital tissues suggests that the disease is autoimmune in nature.16 The major cellular constituents are T lymphocytes showing an increase in both CD4+ and CD8+ cells, with a slight predominance of the latter. In early GO, cell-mediated T helper cell type 1 cells predominate and produce the cytokines interleukin-2 (IL-2), interferon-gamma (IFN-γ), and tumor necrosis factor-α (TNF-α). In more long-standing disease, T helper type 2 cells that participate in humoral responses are dominant and produce IL-4, IL-5, and IL-10.17 The resident macrophages and fibroblasts release additional inflammatory mediators including IL-1α, IL-6, IL-8, IL-16, tumor growth factor-β (TGF-ß), RANTES, and prostaglandin 2 (PGE2). These inflammatory mediators and chemokines incite local inflammation and activate T-cell migration across primed endothelium (Box 56.2).18,19 The stimulatory effects of proinflammatory cytokines result in high production of

A

B

Figure 56.1  (A, B) Patients with severe Graves’ ophthalmopathy demonstrating proptosis, lid retraction, conjunctival erythema, and periorbital edema.

Box 56.1  Clinical background of Graves’

ophthalmology

•Graves’ hyperthyroidism is caused by stimulation of thyroidstimulating hormone (TSH) receptor by TSH receptor autoantibodies

•Approximately 25–50% of patients with Graves’ disease have clinical eye involvement

•The majority of Graves’ ophthalmology patients have an increase in both the orbital fat and extraocular muscle volumes

Box 56.2  Pathology of Graves’ ophthalmology

•The expanded orbital fat compartment in Graves’ ophthalmology contains excess glycosaminoglycans and a diffuse infiltration of lymphocytes

•Orbital fibroblasts produce glycosaminoglycans and a subset have the ability to differentiate into mature adipocytes

•Orbital fibroblasts are thought to be the autoimmune target in Graves’ ophthalmology

hyaluronan by orbital fibroblasts20; a 50% increase in hyaluronan production has been demonstrated in these cells following exposure to IFN-γ.21

Etiology

Genetic contributions

Several genes, including various human leukocyte antigen (HLA) alleles,22–25 cytotoxic T-lymphocyte antigen 4 (CTLA4),26 T-cell receptor (TCR) ß-chain,27 and immunoglobulin heavy chain, confer susceptibility to GD with low relative risk. However, while a number of candidate genes have been studied, including some HLA alleles, TNF-ß,

CTLA4, and TSHR, none has been shown to be associated with the development of GO in patients with GD. This suggests that environmental factors may play a more significant role than genetic factors in the development of the ocular manifestations of GD.11

Mechanical factors and trauma

The signs and symptoms of GO are attributable to mechanical pressures within the noncompliant, unyielding bony orbit owing to the presence of expanded orbital fat and increased extraocular muscle volume. The increased orbital tissue volume within the confines of the bony orbit leads to proptosis, or anterior displacement of the globe, which may be seen as a type of “natural” orbital decompression. The limited space for volume expansion within the bony orbit may impair venous and lymphatic outflow and result in chemosis and periorbital edema.7 Individual variations in the orbital contour or the vascular vessels may make some patients with GD more prone to the development of clinically significant GO.6

The trauma delivered by orbital tissue expansion within a noncompliant bony orbit may aggravate the underlying inflammatory process. This could further stimulate release of proinflammatory cytokines and chemokines and lead to increased presentation of antigen within the orbit and augmentation of the autoimmune response.28

Tobacco smoking

Smoking is the primary risk factor known for the development of GO in patients with GD. The odds ratio, relative to controls, has been reported to be as high as 20.2 for current smokers and 8.9 for ex-smokers, suggesting a direct and immediate effect of smoking.29,30 Smoking is highly associated with more severe GO31 with failure of immunosuppressive therapy,32 and with worsening of GO after radioiodine treatment.

Mechanisms underlying this association between smoking and GO are unclear. Smoking has been associated with other autoimmune diseases, such as rheumatoid arthritis33 and Crohn’s disease,34 suggesting that the autoimmune process may be activated in smokers. However, circulating levels of cytokines do not appear to differ between smokers and nonsmokers, except for the presence of higher levels of IL-6 receptor in the former.35 Patients with both Graves’ hyperthyroidism and GO have higher levels of circulating IL-6 receptor than do hyperthyroid GD patients without clinical GO.36 In spite of this, there appears to be no difference in IL-6 receptor concentrations in the sera of smoking and nonsmoking GO patients.37 In vitro studies have demonstrated that orbital fibroblasts cultured under hypoxic conditions produce increased amounts of hyaluronic acid.38 In addition, exposure to cigarette smoke extract appears to stimulate both adipogenesis and secretion of hyaluronic acid by orbital fibroblasts.39

Radioiodine therapy for Graves’ disease

Studies have suggested that treatment with radioiodine may worsen eye manifestations in patients with GD who have pre-existing, active GO.40–42 In one study, 443 patients with

GO were prospectively treated with radioiodine, methimazole, or the combination of radioiodine and prednisone.40 The percentages of smokers and patients having GO at baseline in each group were comparable. Within 6 months of treatment, mild progression of ocular disease was seen in 15% of patients treated with radioiodine alone, in 2.7% of patients treated with methimazole, and in none of the patients treated with the combination of radioiodine and prednisone. Patients who experienced progression of ocular disease after receiving radioiodine were most commonly smokers and individuals having existing active GO.40 It appears that progression of GO owing to radioiodine therapy does occur in some patients. However, it is generally mild and can be prevented by concurrent administration of corticosteroids. Patients with pre-existing eye disease, or those who smoke or have severe thyrotoxicosis, appear to be more likely to experience this complication (Box 56.3). In addition, ocular disease progression was not seen in another study in which postradioiodine hypothyroidism was prevented, suggesting that hypothyroidism itself might play a role and should be avoided.43 Mechanisms responsible for ocular disease progression following radioiodine are unclear but might include increased TSHR autoantibody production, release of autoantigen from the thyroid, or destruction of radiosensitive suppressor T cells within the thyroid.

Pathophysiology

Orbital fibroblasts

Orbital fibroblasts are phenotypically heterogeneous multipotent cells that can be divided into subpopulations even within a single tissue. These subpopulations are distinguishable on the basis of the cell surface marker Thy-1.6 A minority of fibroblasts originating from the orbital adipose/ connective tissue compartment do not express this antigen (Thy-1–) and are “preadipocyte fibroblasts” capable of differentiating into adipocytes in the presence of PPAR-γ agonist (Figure 56.2). In contrast, perimysial fibroblasts uniformly express Thy-1 (Thy-1+) and lack the ability to undergo adipogenesis. Differences in orbital fibroblast phenotype and the distribution of these cells with orbital compartments may help to explain why some GO patients have predominant eye muscle disease while others have increased orbital adipose tissue volume as the predominant feature.44

Orbital fibroblasts from patients with GO appear to display amplified responses to proinflammatory cytokines compared with fibroblasts from other anatomic sites.11 For

Figure 56.2  Cultured orbital fibroblasts following 10-day exposure to the PPAR-γ agonist rosiglitazone. Adipogenesis is evidenced by oil red O staining of a mature adipocyte.

Box 56.4  Pathophysiology of Graves’

ophthalmology (GO)

•GO orbital fibroblasts display amplified responses to proinflammatory cytokines

•GO orbital fibroblasts are capable of initiating lymphocyte recruitment through their secretion of proinflammatory cytokines

•Cytokine-induced responses with potential relevance to GO

include the inhibition of adipogenesis in orbital fibroblasts by transforming growth factor-β, interferon-γ, and tumor necrosis factor-α and its promotion by interleukin-6

example, while orbital fibroblasts treated with IFN-γ or leukoregulin produce high levels of hyaluronic acid, dermal fibroblasts are only modestly stimulated by these same factors.47 In addition, orbital fibroblasts are capable of initiating lymphocyte recruitment via their secretion of proinflammatory cytokines which, in turn, stimulate the production of chemoattractant molecules by T cells. Two such molecules, IL-16 and RANTES, account for more than 90% of T lymphocyte migration initiated by orbital fibroblasts. This suggests that these two chemoattractants act as important molecular triggers within the orbit, directing lymphocytes to areas of tissue injury and repair. Other cytokineinduced responses with potential relevance to GO include the inhibition of adipogenesis in orbital fibroblasts by TGF- ß, IFN-γ, and TNF-α, and its promotion by IL-6.45,46 Orbital fibroblasts not only respond to cytokines, but also produce cytokines, including IL-1, IL-6, and IL-8, as well as prosta­ glandins, in response to ligation of their CD40 receptors by CD154 on T cells (Box 56.4).48–50

Orbital autoantibodies

TSHR autoantibodies

It is well accepted that autoantibodies directed against the thyroidal TSHR (TRAb) are responsible for Graves’

hyperthyroidism. Due to the close clinical and temporal associations between Graves’ hyperthyroidism and GO, it has been hypothesized that TRAb directed against TSHR expressed in the orbit may underlie the pathogenesis of GO. Indeed, the occurrence of GO is greatest in GD patients with the highest levels of circulating TRAbs,51 and the clinical activity of the disease correlates with serum levels of these autoantibodies.52

A qualifying requirement to link TRAbs to the pathogenesis of GO is that the TSHR be expressed in orbital tissues. Recent studies have shown this to be the case as TSHR mRNA and protein have been demonstrated both in GO orbital adipose tissue and in normal orbital adipose tissue speci- mens.52–54 Further, TSHR expression levels are higher in orbital fat tissues from GO patients than in those from normal individuals.55 Additional support for this hypothesis lies in the positive correlation found between patients’ clinical activity scores and TSHR mRNA levels measured in their orbital tissues removed during decompression surgery.56

Studies in vitro show that orbital preadipocyte fibroblasts can be induced to differentiate into mature adipocytes, and in doing so increase their expression of TSHR. When cultured in the presence in adipogenic PPAR-γ agonists, levels of TSHR mRNA, as well as mRNA encoding various adipocyte-associated genes, increase roughly 10-fold in these cells.57,58 Studies of uncultured orbital fat specimens obtained from GO patients also show high levels of these genes compared with normal orbital fat specimens, suggesting that adipogenesis is increased within the orbit in GO.59 Whether this is directly caused by TRAb targeting TSHR on these cells is an area of active investigation.

Gene array studies comparing GO and normal orbital adipose tissues also reveal increased expression of adipocyterelated genes in GO tissues. In addition, these studies demonstrate high expression of secreted frizzled-related protein-1 (sFRP-1), a known inhibitor of wingless type (Wnt) signaling (Box 56.5).60 As active Wnt signaling inhibits adipogenesis, it is possible that sFRP-1 acts within the GO orbit to turn off this signaling system and thereby stimulate adipogenesis within these tissues.

IGF-1 receptor autoantibodies

Recent studies have implicated insulin-like growth factor-1 receptor (IGF-1R) as another important autoantigen in GO. Evidence for this includes that human orbital fibroblasts display high-affinity IGF-1 binding sites and that immunoglobulin G (IgG) extracted from the sera of GD patients

Box 56.5  Autoantibodies in Graves’

ophthalmology (GO)

•Orbital preadipocyte fibroblasts can be induced to differentiate into mature adipocytes with increased expression of thyroid-stimulating hormone receptors

•Gene array studies reveal increased expression of adipocyterelated genes in GO orbital tissues

•High expression of secreted frizzled-related protein-1 (sFRP-1), a known inhibitor of wingless type (Wnt) signaling, may stimulate adipogenesis in GO orbit

interacts with these sites.61 No such effect was seen using IgG of normal patients. GD IgG has been shown to stimulate orbital fibroblasts to produce the chemokines IL-16 and RANTES, to increase production of cytokines, and to augment their synthesis of hyaluronic acid.62,63 Evidence has been presented that these effects may be mediated through the IGF-1R, suggesting a possible role for autoantibodies directed against this receptor in the pathogenesis of GO.61–63

Novel approaches to therapy

Current medical management of GO is inadequate, generally imparting only modest benefit to patients as they await spontaneous improvement in the disease. While orbital decompression surgery has proven beneficial to patients with severe disease,64 the prevention of this intervention is the goal of medical management. Recent progress in the understanding of GO pathogenesis has led to the identification of several potential targets for novel therapeutic agents (Figure 56.3). As discussed above, both the production of autoantibodies by B cells and the release of proinflammatory cytokines by activated T cells play a role in the development of GO. Therefore, targeting the initial stages of both B- and T-cell activation using costimulation inhibitors (CTLA4-Ig or alefacept) could be of therapeutic benefit (Box 56.6).65

Box 56.6  Novel approaches to therapy of Graves’

ophthalmology (GO)

•Current medical management of GO is inadequate

•Specific monoclonal antibodies and pharmaceuticals aimed at the initial stages of both B- and T-cell activation and adipogenesis may be of therapeutic benefit

•Results of uncontrolled trials of rituximab in the treatment of GO are promising and have paved the way for randomized controlled trials which are now under way

Orbital fibroblast

4

Adipogenesis

Figure 56.3  Potential targets for novel therapeutic agents in Graves’ ophthalmopathy. (1) Costimulation inhibitors (CTLA4-Ig, alefacept); (2) anti-B-cell monoclonal antibody (rituximab); (3) specific novel anti- insulin-like growth factor-1 receptor (anti-IGF-1R) and anti-thyroid- stimulating hormone receptor (anti-TSHR) antibodies; (4) inhibitors of early phases of adipogenesis (PPAR-γ antagonists); and (5) biological agents that block tumor necrosis factor-α (TNF-α) (infliximab, adalimumab, etanercept) or interleukin-1R (anakinra).