The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs) Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer © 2015 Wiley Periodicals, Inc

The Fibroblast Growth Factor signaling pathway

The results of many studies in a variety of species have significantly advanced our understanding of the role of visual experience and the mechanisms of postnatal eye growth, and the development of myopia. This paper surveys and reviews the major contributions that experimental studies using animal models have made to our thinking about emmetropization and development of myopia. These studies established important concepts informing our knowledge of the visual regulation of eye growth and refractive development and have transformed treatment strategies for myopia. Several major findings have come from studies of experimental animal models. These include the eye's ability to detect the sign of retinal defocus and undergo compensatory growth, the local retinal control of eye growth, regulatory changes in choroidal thickness, and the identification of components in the biochemistry of eye growth leading to the characterization of signal cascades regulating eye growth and refractive state. Several of these findings provided the proofs of concepts that form the scientific basis of new and effective clinical treatments for controlling myopia progression in humans. Experimental animal models continue to provide new insights into the cellular and molecular mechanisms of eye growth control, including the identification of potential new targets for drug development and future treatments needed to stem the increasing prevalence of myopia and the vision-threatening conditions associated with this disease.

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IMI - Report on Experimental Models of Emmetropization and Myopia.

Circadian rhythms, refractive development, and myopia.

FGF10: A Multifunctional Mesenchymal-Epithelial Signaling Growth Factor in Development, Health, and Disease

Refractive errors in vision can be caused by aberrant axial length of the eye, irregular corneal shape, or lens abnormalities. Causes of eye length overgrowth include multiple genetic loci, and visual parameters. We evaluate zebrafish as a potential animal model for studies of the genetic, cellular, and signaling basis of emmetropization and myopia. Axial length and other eye dimensions of zebrafish were measured using spectral domain-optical coherence tomography (SD-OCT). We used ocular lens and body metrics to normalize and compare eye size and relative refractive error (difference between observed retinal radial length and controls) in wild-type and lrp2 zebrafish. Zebrafish were dark-reared to assess effects of visual deprivation on eye size. Two relative measurements, ocular axial length to body length and axial length to lens diameter, were found to accurately normalize comparisons of eye sizes between different sized fish (R2=0.9548, R2=0.9921). Ray-traced focal lengths of wild-type zebrafish lenses were equal to their retinal radii, while lrp2 eyes had longer retinal radii than focal lengths. Both genetic mutation (lrp2) and environmental manipulation (dark-rearing) caused elongated eye axes. lrp2 mutants had relative refractive errors of -0.327 compared to wild-types, and dark-reared wild-type fish had relative refractive errors of -0.132 compared to light-reared siblings. Therefore, zebrafish eye anatomy (axial length, lens radius, retinal radius) can be rapidly and accurately measured by SD-OCT, facilitating longitudinal studies of regulated eye growth and emmetropization. Specifically, genes homologous to human myopia candidates may be modified, inactivated or overexpressed in zebrafish, and myopia-sensitizing conditions used to probe gene-environment interactions. Our studies provide foundation for such investigations into genetic contributions that control eye size and impact refractive errors.

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Rapid, accurate, and non-invasive measurement of zebrafish axial length and other eye dimensions using SD-OCT allows longitudinal analysis of myopia and emmetropization.

Purpose Fibroblast growth factor-10 (FGF10) can modulate extracellular matrix associated genes and, therefore, it could be a myopia susceptibility gene. This study used an animal model, single nucleotide polymorphisms (SNPs) association, and genetic functional assay to evaluate FGF10 gene for myopia. Methods The expression levels of FGF10 gene were compared among the form deprivation myopic (FDM) eyes, the fellow eyes of the FDM group, and the healthy eyes of experimental mice. In the present study 1020 cases (≤-6.0 diopters [D]) and 960 controls (≥-1.5 D) were enrolled from a Chinese population. Eight tagging SNPs were genotyped to test for an association between genotypes and myopia. The luciferase reporter assay was conducted for the particular SNP to assess the allelic effect on gene expression. Results The sclera of FDM eyes had a 2.57-fold higher level of FGF10 mRNA (P = 0.018) than the fellow eyes. Although no SNP was associated with high myopia, SNP rs339501 was significantly associated with extreme myopia (≤-10 D, P = 0.008) and the odds ratio (OR) was 1.58 for G allele carriers. The luciferase assay showed that the risk G allele significantly caused a higher expression level than the A allele (P = 0.011). Conclusions The evidence suggested FGF10 to be a risk factor for myopia. The sclera of myopic eyes had higher FGF10 levels. The risk G allele of SNP rs339501 was associated with extreme myopia in human and caused a higher gene expression in the luciferase assay. It is concluded that the FGF10 could have been involved in the development of myopia.

A functional polymorphism at the FGF10 gene is associated with extreme myopia.

AIM To test for the association between genome-wide methylation and myopia in human and mice. METHODS Long interspersed nucleotide element 1 (LINE-1) methylation levels were used to surrogate genome-wide methylation level. We first tested for the association between high myopia (<-6 D) and LINE-1 methylation in leukocytes in 220 cases and 220 control subjects. Secondly, we validated the results of LINE-1 methylation in eyes from the form deprivation myopia (FDM) mice. Furthermore, we calculated the correlation of LINE-1 methylation levels between leukocyte DNA and ocular DNA in the mice. We also tested whether dopamine can alter LINE-1 methylation levels. RESULTS The LINE-1 methylation level was significantly higher in the myopic human subjects than controls. The upper and middle tertiles of the methylation levels increased an approximately 2-fold (P≤0.002) risk for myopia than the lower tertile. Similarly, FDM mice had high LINE-1 methylation levels in the leukocyte, retina and sclera, and furthermore the methylation levels detected from these three tissues were significantly correlated. Immunohistochemical staining revealed higher levels of homocysteine and methionine in the rodent myopic eyes than normal eyes. Dopamine treatment to the cells reduced both LINE-1 methylation and DNA methyltransferase levels. CONCLUSION LINE-1 hypermethylation may be associated with high myopia in human and mice. Homocysteine and methionine are accumulated in myopic eyes, which may provide excess methyl group for genome-wide methylation.

Genome-wide DNA hypermethylation and homocysteine increase a risk for myopia.

Purpose: Fibroblast growth factor-2 (FGF2) has been implied in the development of myopia according to previous studies investigating FGF2 in the sclera and retinal pigment epithelium. This study measured retinal FGF2 gene expression in an animal model and also tested for the association between single nucleotide polymorphisms (SNPs) in FGF2 and high myopia. Methods: The guinea pigs were assigned to 2 groups: form deprivation myopia (FDM) for two weeks and normal control (free of form deprivation). Biometric measurement was performed and FGF2 expression levels were compared among the FDM eyes, the fellow eyes of the FDM group and the normal eyes in retina. We also enrolled 1,064 cases (≤-6.0 D) and 1,001 controls (≥-1.5 D) from a Chinese population residing in Taiwan. Six tagging SNPs were genotyped to test for an association between genotypes and high myopia. Results: The FDM eyes had the most prominent changes of refraction and axial length. Compared with the mRNA levels of FGF2 in the normal eyes, the FDM eyes had the highest levels of mRNA (p=0.0004) followed by the fellow eyes (p=0.002). The FDM and normal eyes became more myopic compared with the fellow eyes, but the fellow eyes became more hyperopic (p=0.004) in the end of the experiment which may be due to its relatively short axial length when compared with normal eyes (p=0.05). The SNP genotypes were all in Hardy–Weinberg equilibrium. However, none of the SNPs were significantly associated with high myopia (all p values >0.1). Conclusions: We identified a significant change of FGF2 expression in the FDM eyes but FGF2 genetic variants are unlikely to influence susceptibility to myopia. There may be a systemic effect to influence gene expression and refraction on the fellow eyes, which may perturb emmetropization in the fellow eyes. Our data also suggest using normal eyes rather than the fellow eyes as the control eyes when study the form deprivation myopia.

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The FGF2 gene in a myopia animal model and human subjects

Purpose: We tested the role of microRNA-328 in dry eye disease (DED). Benzalkonium chloride (BAC) has been used to induce DED in animal models. We first demonstrated that both BAC and hyperosmotic stress induced overexpression of miR-328 in corneal cells and then tested whether anti-miR-328 could be a new therapy. Methods: BAC was instilled to both eyes of 41 rabbits and 19 mice from day 0 to 21 to induce DED. Animals of each species were divided to receive topical instillation of saline or anti-miR-328 eye drops between day 8 and 21. The DED signs were assessed by corneal fluorescein staining, histological examination, apoptosis of corneal cells, and inflammatory cytokines in rabbit eyes. For mice, only corneal fluorescein staining was assessed for the therapeutic effects. The corneal fluorescein staining scores ranged from 0 of no staining to 4 of coalescent. Results: For the rabbits, the staining score was significantly reduced (P = 0.038) after the 14-day anti-miR-328 treatment (n = 42 eyes), but the score was not improved by saline treatment (n = 40 eyes). Furthermore, rabbit eyes treated with anti-miR-328 had thicker corneal epithelium (P = 9.4 × 10-5), fewer apoptotic cells in corneal epithelium (P = 0.002), and stroma (P = 0.029) compared with the saline-treated eyes. Anti-miR-328 was more effective than saline to reduce the block of orifices of Meibomian glands, although such an effect was only marginally significant (P = 0.059). Similarly, anti-miR-328 was more effective than saline in reducing corneal staining in mouse eyes (P = 0.005). Conclusion: Overexpression of miR-328 may contribute to DED. Anti-miR-328 protects corneal cells and promotes re-epithelialization for DED treatment. 

MicroRNA Therapy for Dry Eye Disease

The present invention is directed to anti-sense microRNA-328 in a form of oligodeoxyribonucleotides, or locked nucleic acid (LNA)-modified, and phosphorothioated (PS) bond-modified oligonucleotides. The present invention is also directed to a pharmaceutical composition comprising the anti-sense microRNA-328 composition and a pharmaceutically acceptable carrier. The present invention is further directed to a method for preventing or treating myopia by administering to a subject the anti-sense microRNA-328 composition. A preferred route of administration is topical administration to the eyes.

Chung-Ling Liang

Papers

A functional polymorphism at the FGF10 gene is associated with extreme myopia.

Genome-wide DNA hypermethylation and homocysteine increase a risk for myopia.

The FGF2 gene in a myopia animal model and human subjects

MicroRNA Therapy for Dry Eye Disease

MicroRNA-328 anti-sense composition and therapeutic use