Scleral remodeling in early adulthood: the role of FGF-2

Emmetropization, a natural process of ocular elongation, is closely associated with scleral remodeling. The Fibroblast growth factor-2 (FGF-2) was reported involved in scleral remodeling in myopia models. Herein, we aimed to investigate the role of scleral fibroblast-to-myofibroblast differentiation and FGF-2 in scleral remodeling during maturation. Our findings revealed that the posterior scleral fibroblasts (SFs) from mature guinea pigs exhibit increased stiffness compared to those from young guinea pigs. Moreover, mature SFs displayed decreased cell proliferation but increased levels of α-SMA, matrix metalloproteinase 2 (MMP2), and collagen 1, when compared to young SFs. Additionally, the mRNA expression of scleral Fgf-2, Fgf receptor 1 (Fgfr1), Fgfr2, Fgfr3, and Fgfr4 was increased in mature SFs. Notably, exogenous FGF-2 showed increased cell proliferation and led to decreased expression of α-SMA, MMP2, and collagen 1 in mature SFs. Overall, our findings highlight the influence of maturation on SFs from posterior scleral shells, resulting in increased stiffness and the manifestation of fibroblast-to-myofibroblast differentiation during development. Exogenous FGF-2 increased cell proliferation and reversed the age-related fibroblast-to-myofibroblast differentiation, suggesting a potential role of FGF-2 in regulating scleral remodeling.


Results
Posterior SF stiffness increased with sclera maturation Previous studies have indicated that age is associated with the stiffness of sclera tissue 11 .To further clarified of changes in the biomechanical characteristic of the sclera, we quantitatively analyzed the biomechanical change and obtained morphological data in the living SFs using atomic force microscopy (AFM) (Fig. 1A-C).The Young's modulus was significantly higher in the posterior SFs of mature guinea pigs than in those of the young guinea pigs (11.95 ± 2.24 kPa vs. 9.76 ± 1.90 kPa, P < 0.01) (Fig. 1D).Meanwhile, the Young's modulus did not show statistical significance when comparing the equatorial SFs of the mature guinea pigs to those of the young guinea pigs (9.69 ± 2.61 kPa vs. 9.36 ± 1.82 kPa, P > 0.05).Interestingly, the mature SFs from posterior scleral shells were stiffer than the mature SFs from equatorial scleral shells, as evidenced in a significantly higher Young's modulus (11.95 ± 2.24 kPa vs. 9.69 ± 2.61 kPa, P < 0.01).These data demonstrate that the SFs from posterior scleral shells were affected by natural maturation, and the posterior SFs from mature guinea pigs were stiffer than those from young guinea pigs.Given this finding, the following study used only SFs from posterior scleral shells.

SFs exhibit myofibroblast transition during sclera maturation
To further determine whether SFs exhibit myofibroblast transition during sclera maturation, we examined the expression of α-SMA and collagen 1 17 , which are markers of myofibroblast and ECM changes.Increased α-SMA expression in mature SFs was demonstrated using immunofluorescence and western blot (Fig. 2A-C).As shown in Fig. 2B, C, MMP2 and collagen 1 increased in mature guinea pigs' SFs.Cell proliferation marker Ki67 was decreased in mature guinea pigs' SFs.Consistently, young guinea pigs' SFs were more proliferative, as the young group had significantly higher OD values than that of the mature group (1.12 ± 0.009 versus 0.74 ± 0.042 at 24 h, P < 0.001; Fig. 2D).These results indicate that SFs exhibit myofibroblast transition and ECM changes during natural maturation.

Discussion
Fibroblasts are cells that are inherent to the sclera, supporting ocular function and integrity.Around 25% of scleral thickness is occupied by fibroblasts 19 .SFs contribute to scleral remodeling in myopia 3 , however, the role of SFs during scleral tissue maturation is not yet well understood.In this study, we first demonstrated that SFs from posterior scleral shells are affected by natural maturation, becoming stiffer and exhibiting fibroblast-to-myofibroblast differentiation during maturation.Fgf-2 and Fgfrs were upregulated during maturation as well.Exogenous FGF-2 increased cell proliferation and reversed the age-related fibroblast-to-myofibroblast differentiation.
The growth of axial length and the process of achieving emmetropia in guinea pigs exhibit similarities to humans 20 .In humans, axial length increases by 25% during the initial period of 2-3 years post-birth, while only by 1% over the subsequent ten years.In the guinea pig, axial length also increases more during first 3 weeks (12% infancy, 8% juvenile, 2% adolescent) 16,21 .Notably, myopia often occurs during juvenile stage.Therefore, in the present study, juvenile and adult guinea pigs have been selected as experimental subjects to investigate the process of scleral remodeling during emmetropization.
The sclera is an elastic, fibrous tissue, and there are race-and age-related differences in scleral elasticity and stiffness.Compared to the eyes of Europeans, for example, the eyes of Africans have higher shear stiffness and lower amounts of stretch 22 .Measuring the tangent modulus-stress-strain of the sclera demonstrated that the sclera becomes stiffer with aging 11 .In addition, there were regional variations in the biomechanical properties of the sclera.From the posterior to anterior regions of the sclera, extensibility decreased, and the tangent modulus increased 11,23 .However, all these studies used scleral tissue to analyze the biomechanical behavior of the sclera.There is little data on the change of SFs that might have an impact on sclera tissue.Our study appears to be the first to use AFM in the characterization of the biomechanical properties of living SFs in both young and mature guinea pigs and from the posterior to anterior regions of the sclera.We consistently found that SFs from posterior shells were stiffer than SFs from the equator in mature guinea pigs.Meanwhile, the SFs from the posterior shell became stiffer during maturation, while the SFs from the equator did not change.
Protein profile, proteoglycan, and ECM composition change during normal scleral growth 3,10,12 .Collagen makes up 50% of the scleral tissue by weight, with 95% of that collagen being type 1 (namely collagen 1) 24 .Evidence indicates that decreased expression of collagen 1 is responsible for ocular elongation and the thinning of the sclera 25 .Consistent with previous research 26 , scleral collagen 1 synthesis was higher than decomposition during the processes of maturation or emmetropization, since expression of collagen 1 increased in the SFs of mature guinea pigs.The main role of MMP2 is to degrade collagen 1 in the sclera, which can help maintain balance in scleral remodeling 27 .Our study found that the expression of MMP2 was increased, which was also consistent with previous research 26 .We speculated that the expression level of collagen 1 might play a pivotal www.nature.com/scientificreports/role in maintaining scleral remodeling equilibrium, wherein reduced collagen 1 expression levels could serve as a molecular mechanism driving the transition from emmetropia to myopia.Myofibroblasts are enriched with α-SMA, which augments their ability to generate contractile force and increases tissue stiffness 9 .Our results also showed that both the expression of α-SMA and the Young's modulus were increased.Like the changes in SFs related to myopia 8 , SFs also exhibited fibroblast-to-myofibroblast differentiation during maturation, which is characterized by a higher expression of α-SMA.The scleral stiffness increased in a form-deprivation myopia model using tree shrews as well 28 .Although further experiments are needed, we speculate that myopia might be a process of excessive emmetropia.This could be explained by ocular elongation, which is a common feature of both emmetropization and myopia.
FGF-2 is an established growth factor for cell proliferation, ECM remodeling, and inhibition of myopia 18,29,30 .Expression of scleral Fgfr1 was significantly upregulated while endogenous Fgf-2 was not significantly upregulated in myopic eyes 18 .Interestingly, our results first found that both Fgf-2 and Fgfrs were significantly upregulated during maturation.We speculate that the upregulation of endogenous Fgf-2 might help keep the eyes from excessive ocular elongation.Intravitreal application of FGF-2 has been shown to inhibit ocular elongation in the form of myopia deprivation (FDM) in a model using chicks 31 .In an FDM model using guinea pigs, peribulbar injection of FGF-2 also inhibited ocular elongation through upregulating collagen 1, integrin α2, and β1 14 .Our study consistently found that FGF-2 promoted cell proliferation.The FGF-2 regulated ECM by downregulating the expression of α-SMA, MMP2, and collagen 1, indicating inhibition of myofibroblast transition.The expression of collagen 1 resulted in a different type of change than that shown in a previous study, however, considering the different experimental models at play, more experiments are needed to understand this difference.This evidence as a whole indicates that FGF-2 is an effective mediator of controlling scleral remodeling and ocular elongation.
However, we acknowledge the limitations of our research, as we only included guinea pigs that were 4 weeks old and 6 months old in our study.To gain a comprehensive understanding of the changes in the sclera with age and regions, we would consider including guinea pigs of younger and older age groups and SF from different www.nature.com/scientificreports/regions of sclera in future studies.Moreover, while we have evaluated the expression levels of Fgf-2 and Fgfrs in SFs at the RNA level, it would be beneficial to investigate the change in protein levels in subsequent studies.

Conclusion
In conclusion, our study first found that the fibroblast-to-myofibroblast differentiation was a normal part of scleral remodeling during maturation, with SFs from posterior scleral shells becoming stiffer.Both Fgf-2 and Fgfrs were upregulated in mature SFs.Exogenous FGF-2 increased cell proliferation and reversed the age-related fibroblast-to-myofibroblast differentiation indicating that FGF-2 is an important mediator of scleral remodeling and ocular elongation.Although further translational studies are required, our study investigated the important roles of scleral fibroblast-to-myofibroblast differentiation and FGF-2 in the control of scleral remodeling during maturation.

Animals
Healthy guinea pigs were purchased from Jiagan Biotechnology Co. Ltd (Shanghai, China).Pigmented guinea pigs were divided into two groups: young guinea pigs (four weeks old, randomly female and male) and adult guinea pigs (six months old, randomly female and male).They were reared in an animal facility under a 12-h light/dark cycle.Each experiment was repeated at least three times using different guinea pigs.At least 15 young and 15 adult pigmented guinea pigs were used in this study.We used an equal number of males and females in each experiment.Experiments were performed according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.This study protocol (No. 2014-004) was approved by the ethics committee of Zhongshan Ophthalmic Center, Sun-Yet Sen University.And this study is reported in accordance with ARRIVE guidelines.Refractive error and axial length measurements were obtained using streak retinoscopy and A-scan ultrasound (Supplementary Table 1).

Cell isolation and culture
Guinea pigs were euthanized by overdose with an intraperitoneal injection of sodium pentobarbital and their right eyes were taken for further study.A circumferential incision was made along the limbus of the eyeball, the anterior segment of the eye and the lens were discarded, then the vitreous body, retina, and RPE-Bruch's membrane-choroid complex were removed.Afterward, the scleral shells were divided into two regions, the equatorial and posterior sclera (Fig. 6A, B) 32,33 .The explanted tissues were cut into approximately 1 × 1 mm pieces (Fig. 6C), and then placed in 60 mm Nunc™ EasYDish™ (Thermo Fisher Scientific, Waltham, MA, USA) in Dulbecco's modified Eagle's medium (Nutrient Mixture F-12 [DMEM/F12; GIBCO, Grand Island, NY, USA]).Each 60 mm culture dish contained tissues from one animal.The medium was supplemented with a 10% HyClone™ Fetal Bovine Serum (FBS; GE Healthcare Life Sciences Company, Pittsburgh, PA, USA).The growth medium was replaced every 2-3 days.After 5-7 days of incubation at 37 °C and 5% CO 2 in a humidified incubator, cells grew out of the scleral explants.Confirmation of the phenotype of primary cells was obtained by fluorescent immunostaining with vimentin, as described previously 34 .

Atomic force microscopy (AFM)
A Dimension Fastscan with ScanAsyst™ AFM (Bruker, Santa Barbara, CA, USA) was used to analyze the biomechanical differences between young and mature groups of living SFs.The posterior and equatorial scleral shells of the guinea pigs were collected.After cell isolation, SFs were used to take atomic force microscopy measurements.The data were collected in at least six regions of each culture.An area of cells measuring 20 × 20 μm 2 was imaged at a resolution of 128 × 128 pixels in contact mode, with a pyramidal tip of silicon nitride cantilevers (nominal spring constant k = 0.03N/m, MLCT-D, Bruker) selected.This test was performed as previously described 35 .A NanoScope Analysis (Bruker) was used to calculate cell elasticity values.The Young's modulus was calculated using the Hertz model, which was selected from eight loci (500 × 500 nm) on each cell.The test temperature was controlled at 37 °C prior to AFM force indentation.

Cell proliferation assay
Cell counting kit-8 (CCK-8; Dojindo, Shanghai, China) was used to assess cell proliferation.The cells were cultured at a density of 1 × 10 4 cells/well in a 96-well plate with a serum-free medium for 24 h.Cells were then treated with or without 10-50 ng/ml fibroblast growth factor 2 (FGF-2; Peprotech, Rocky Hill, NJ, USA) for another 24 h, 48 h and 72 h.Then, 10 μl CCK-8 solution was added and incubated at 37 °C for 2 h.The optical density (OD) values were detected using a 96-well multiscanner autoreader (Synergy H1, BioTek, Winooski, VT, USA) at 450 nm.

Flow cytometry
Following a three-day-culture, SFs were digested with trypsin for 1 min and harvested.The cells were fixed and permeabilized with Fix/Perm solution (BD Bioscience, NJ, USA) for 15 min at 4 °C.Then the cells were incubated with anti-α-SMA-antibody (1:100; ab184675; Abcam) for 45 min in perm/wash buffer (BD Bioscience).All samples were acquired using BD LSR II (BD Bioscience) and data were analyzed with FlowJo v10 (Tree Star Inc, OR, USA).

Western blot
SFs were scraped and lysed in ice-cold RIPA buffer (Beyotime Biotechnology, Shanghai, China), supplied with protease inhibitor (A32963, Thermo Fisher Scientific).Protein concentrations were determined using the BCA-100 Protein Quantitative Analysis Kit (Biocolor Bioscience & Technology Co., Ltd., Shanghai, China).Equivalent protein amounts (15 μg/lane) were separated on 10% SDS-polyacrylamide gels and electro transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore Corporation, Bedford, MA, USA) using wet blotting.Then the PVDF membranes were blocked in 5% nonfat milk for 1 h.Blots were incubated with specific primary antibodies at 4 °C overnight and washed with PBST (0.1% Tween-20 in phosphate buffered saline).Then the blots were incubated with horseradish peroxidase

Statistical analysis
Statistical analyses were performed using SPSS 19.0 (SPSS, Chicago, IL, USA).Statistical significance was determined with an analysis of variance (ANOVA), followed by Tukey's and Bonferroni post-hoc analysis.The Bonferroni post-hoc test was used when variances were equal, while Tukey's post-hoc test was used when they were not.Because the data were not normally distributed, a nonparametric test (two-sided Wilcoxon rank-sum test) was performed on the AFM measurements.All values were presented as mean ± SD. Results were considered statistically significant if values were P < 0.05.

Figure 6 .
Figure 6.Cartoon schematic of equatorial and posterior sclera.The eyes of guinea pigs were harvested after euthanasia.(A) A circumferential incision was made along the limbus of the eyeball, then the anterior segment of the eye and the lens were discarded.(B) Then, the vitreous body, retina, and RPE-Bruch's membranechoroid complex were removed under a dissecting microscope.The scleral shells were divided into two regions: equatorial and posterior sclera.(C) The explanted tissues were cut and then placed in dishes for cell culture.