Extracellular Matrix Stiffness Modulates Myopia Scleral Remodeling Through Integrin/F-Actin/YAP Axis

Abstract

Purpose: Scleral extracellular matrix (ECM) remodeling and weakened scleral stiffness are characteristic of myopia. The purpose of this study was to investigate the precise underlying mechanisms of scleral remodeling regulated by mechanical signals emanating from the ECM.

Methods: The expression and regulation of YES-associated protein (YAP) were confirmed in human samples or guinea pig myopia models by Western blot (WB) or ELISA. To mimic the biomechanical microenvironment associated with myopia, stiff (50 kPa) and soft (8 kPa) substrates were established. The underlying mechanisms were further investigated by quantitative real-time RT–PCR, WB, and fluorescence staining in cells treated with siRNAs, plasmids or inhibitors. In vivo, a YAP activator, inhibitor and F-actin polymerization facilitator were applied to evaluate their therapeutic significance for myopia.

Results: Our findings revealed that YAP expression is decreased in the sclera of guinea pigs and humans with myopia. Under mechanical stimuli, YAP functions as a mediator, transducing mechanical signals and modulating collagen expression. Furthermore, integrin α1β1 acts as a regulator of YAP and operates through modification of the F-actin cytoskeleton. Specifically, in response to mechanical forces, integrin α1β1 modulates F-actin restructuring. This modified actin cytoskeletal architecture subsequently facilitates the nuclear translocation of YAP, ultimately leading to the suppression of COL1A1 expression.

Conclusions: Our results suggest that the integrin α1β1–F-actin–YAP–COL1A1 axis constitutes a vital regulatory mechanism intrinsically associated with the pathogenesis of myopia.

Myopia, the most prevalent refractive error worldwide, is responsible for irreversible visual impairment.¹ Its primary characteristic is excessive elongation of the ocular globe.² The sclera, a dense and resilient connective tissue that forms the outer coat of the eye, undergoes significant changes in myopia. Extensive evidence from clinical and experimental studies has confirmed that axial elongation in myopia is associated with remodeling of the sclera's extracellular matrix (ECM).³ This remodeling process, which is characterized by reduced collagen content, is a leading cause of decreased elasticity in myopic eyes, ultimately resulting in reduced scleral resistance to expansion caused by intraocular pressure.^4,5 The theory of stress-mediated growth and remodeling suggests that mechanical cues from the ECM play a crucial role in controlling cell behavior and gene expression.^6,7 However, the impact of these mechanical changes on the function of scleral fibroblasts and the process of myopic scleral remodeling remains unclear. 

YES-associated protein (YAP) is a transcription coactivator within the canonical Hippo signaling pathway that has recently been acknowledged as a critical mediator of cellular responses to ECM mechanical cues.⁸ YAP has the ability to move between the cytoplasm and the nucleus, where it interacts with DNA-binding transcription factors and regulates gene expression.⁹ The localization of YAP is influenced by external mechanical cues, such as ECM stiffness and fluid shear stress, which are in turn dependent on the internal tension of the cytoskeleton.^10,11 When subjected to mechanical stimuli, the actin cytoskeleton is able to detect and respond to both internally and externally generated forces, subsequently activating YAP.¹² A previous study demonstrated that knocking down the actin capping protein resulted in the formation of F-actin stress fibers and the nuclear localization of YAP.¹³ Conversely, the depletion of F-actin causes YAP to accumulate in the cytoplasm.¹⁴ These findings suggest a hierarchical mechanism in which YAP acts as a downstream sensor of ECM stiffness. Our previous research has uncovered a decrease in ECM stiffness in the sclera during myopia development.¹⁵ However, it remains unknown whether YAP is involved in scleral remodeling, and the impact of ECM stiffness on YAP through the integrin-cytoskeleton axis, as well as its role in regulating scleral remodeling, remain unclear. 

In the present study, we identified integrin α1β1 as a regulator of YAP, which is induced by mechanical stimuli and depends on the actin cytoskeleton. Mechanistically, integrin α1β1 may function as a sensor of matrix stiffness, leading to F-actin reorganization, YAP translocation to the nucleus, and decreased expression of COL1A1 in scleral fibroblasts. Overall, integrin α1β1 plays a vital role in bridging the gap between ECM stiffness and YAP-mediated transcriptional regulation in scleral remodeling. This research provides insight into the involvement of F-actin and integrin α1β1 in the activation of YAP controlled by ECM stiffness, representing a significant advancement in our understanding of the mechanisms behind the regulation of mechanical forces in myopia scleral remodeling. 

Methods

Form-Deprivation (FD) Myopia Model in Guinea Pigs

All procedures were conducted according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. The research protocols were approved by the ethics committee of the Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine (2023AWS113). Two-week-old pigmented guinea pigs were raised at the Laboratory Animal Center under a 12-hour light/dark cycle. They were then monocularly deprived of vision by covering one eye for four weeks with a translucent diffuser to induce FD myopia, whereas the other eye served as the untreated control. 

Refraction and axial length (AXL) in both eyes were measured at baseline and every week during FD treatment. Refractive status data were collected by an automated infrared photorefractor and analyzed through an average of 50 to 100 consecutive measurements via custom software designed by Frank Schaeffel in a dark environment as described previously. AXL was determined using A-scan ultrasonography (Strong 6000A; Wuhan Strong Electronics Ltd., Wuhan, China) with ocular surface anesthesia (0.4% oxybuprocaine hydrochloride). At least eight repeated measurements were averaged for each eye. 

Human Sclera Tissue

Eight eyes from human donors (35 to 70 years old) were obtained from the Eye Bank of Shanghai General Hospital (Shanghai, China). The use of human tissues was in compliance with the tenets of the Declaration of Helsinki and was approved by the Medical Ethics Committee of Shanghai General Hospital (2023SQ086). All of the donors were Chinese, with six males and two females. The donor eyes were obtained as the posterior poles with the cornea removed. The refraction was obtained from the next-of-kin questionnaire. The AXL was measured via A-scan. The detailed information is presented in Supplementary Table S1. The statistical analysis of the characteristics of the donor eyes is presented in Supplementary Table S2. 

Participant Recruitment and Serum Connective Tissue Growth Factor (CTGF) Measurement

Participants were recruited from Shanghai General Hospital Eye Center with written informed consent according to the tenets of the Helsinki Declaration. All participants received noncycloplegic autorefraction of both eyes. Refraction errors are presented as spherical equivalent (SE). Data from the right eye of each patient were included in the analysis. Those with mild myopia (−3.0D ≤ SE < −0.5D) were set as controls, whereas those in the high myopia group had a refraction greater than −6.0D. The exclusion criteria included a history of ocular surgeries; the presence of active inflammatory diseases and congenital disorders in the eyes; glaucoma; diabetes; hypertension; hyperlipemia; liver or renal disorders; autoimmune diseases; and malignancies. This clinical study was approved by the Medical Ethics Committee of Shanghai General Hospital (2022KY038). 

Finally, 76 eligible participants underwent comprehensive ophthalmic examinations. The detailed information is presented in Supplementary Table S3. Blood samples were collected in the morning while the subjects were fasting and centrifuged at 1500 rpm for 10 minutes to obtain the serum. The samples were subsequently stored at −80°C for further analysis. Serum connective tissue growth factor (CTGF) levels were detected using a human ELISA kit (Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. Tests were performed in duplicate, and the mean value was calculated. 

Primary Culture of Scleral Fibroblasts

Primary scleral fibroblasts were extracted from scleral explants of two-week-old guinea pigs. Scleral tissue was cut into blocks of 1 × 1 × 1 mm³ under sterile conditions and carefully placed into separate dishes in Dulbecco's modified Eagle's medium (DMEM; high glucose, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific). Fibroblasts were incubated at 37°C in a humidified atmosphere containing 5% CO₂ until confluent. The medium was replenished twice a week, and the cells at 80% confluence were passaged by using 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific). Fibroblasts from the third and sixth passages were used for experiments after cell identification with appropriate markers, such as vimentin (+) and keratin (−). 

In Vitro Cell Culture on Substrates With Different Stiffnesses

Scleral fibroblasts were seeded at 5 × 10⁴/mL on either stiff (50 kPa) or soft (8 kPa) polyacrylamide hydrogels coated with collagen I in 6-well plates or 24-well plates (Softwell; Matrigen, Irvine, CA, USA) and placed in an incubator at 37°C for 12 hours. Before the experiments, the fibroblasts were washed three times with PBS. In the in vitro intervention experiment, siYAP, siItgα1, cytochalasin D (cytoD, an F-actin depolymerizer), verteporfin (VP, a YAP inhibitor) were used to inhibit the integrin–F-actin–YAP signaling pathway, and YAP plasmid jasplakinolide (an F-actin polymerization facilitator) were used to activate this pathway. Cells transfected with siRNA or plasmid were seeded directly. For the drug-administered group, the cells were first seeded into six-well plates or 24-well plates with different matrix stiffnesses. After three hours, jasplakinolide (25 nM), cytochalasin D (0.5 µM), and verteporfin (250 nM) were added to one of the wells, and the cells were incubated for an additional 12 hours. Concentrations were determined on the basis of minimal changes in cell viability and cell architecture; a shrunken, crumpled appearance should not be observed during culture. 

SiRNAs and YAP Plasmid Transfection

SiRNAs against integrin α1 and YAP were synthesized by GenePharma (Shanghai, China). The sequences of the siRNAs are listed in Supplementary Table S4. In brief, fibroblasts were cultured in six-well plates until they reached 30–50% confluence. siRNAs were transfected into cells using siRNA-Mate (GenePharma) reagent in one well of six-well plates according to the manufacturer's instructions. After 48 h of transfection, the medium was changed to fresh 10% FBS-loaded DMEM for verification of the protein knockdown efficiency or further experiments. 

Plasmids expressing YAP-specific mRNA or vector control plasmids were also constructed by GenePharma. Scleral fibroblasts were transfected with plasmids via the Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA). After incubation for 48 hours at 37 °C, the transfection efficiency of the plasmids was assessed by the fluorescence intensity of green fluorescent protein (GFP) expression and the protein expression of YAP. 

In Vivo Intervention of F-Actin and YAP via Subconjunctival Injection

To examine the effect of YAP signaling on myopia development in vivo, guinea pigs were randomly divided into four groups: (1) the FD + dimethylsulfoxide control group; (2) the FD + verteporfin (700 nM × 100 µL/eye) group; (3) the FD + xmu-mp-1 (10 µM × 100 µL/eye) group; and (4) the FD + jasplakinolide (25 nM × 100 µL/eye) group. Drugs were dissolved in dimethylsulfoxide. Each guinea pig received unilateral subconjunctival injections of these drugs for four weeks to reach a balance of maximizing effectiveness and avoiding intraocular damage. Jasplakinolide and verteporfin were given once a week, and xmu-mp-1 was given three times a week. Topical anesthesia was administered with one drop of 0.4% oxybuprocaine hydrochloride before injection, and levofloxacin was administered to prevent infection. Overall, the injection duration did not exceed 30 seconds, and then the translucent diffuser used for FD was immediately reset over the eye. 

Quantitative RT-PCR (qRT-PCR)

Total RNA from cells and tissues was harvested with TRIzol reagent (Invitrogen) and isolated as described by the manufacturer's specifications. Reverse transcription was performed using the PrimeScript RT reagent kit (Takara Bio, Japan). Quantitative RT-PCR (qRT-PCR) was performed with SYBR Premix Ex Taq II (Takara Biotechnology Co., Kyoto, Japan). An applied Biosystems ViiA 7 system (Life Technologies, Carlsbad, CA, USA) was used for detection. The sequences of the primers used in this experiment are shown in Supplementary Table S4. The cycle threshold (Ct) values were analyzed using the 2^−ΔΔCt method, and the target mRNA levels were normalized to those of GAPDH. 

WB Analysis

The scleral tissues or cultured fibroblasts were lysed in RIPA buffer (Beyotime Biotechnology, Jiangsu, China) supplemented with protease and phosphatase inhibitors. The supernatants were collected after centrifugation. A bicinchoninic acid protein assay was used to determine the protein concentration. Proteins were separated by 10% SDS‒PAGE and transferred to PVDF transfer membranes (Millipore Corporation, Temecula, CA, USA). The membranes were incubated in 5% milk/TBST, followed by overnight incubation at 4°C with the corresponding primary antibodies against YAP (1:1000, no. 14074; Cell Signaling Technology, Danvers, MA, USA), integrin α1 (1:1000, ab181434; Abcam), integrin β1 (1:1000, ab30394; Abcam), collagen I α1 (1:1000, #72026, CST, USA; 1;1000, ab138492; Abcam), and GAPDH (1:1000, 60004-1-Ig; Proteintech, Wuhan, China). After being rinsed in TBST, the immunoblot was incubated for one hour with horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse IgG (115-005-146, 111-005-003; Jackson ImmunoResearch Labs, West Grove, PA, USA). The images were visualized via enhanced chemiluminescence. At least three independent experiments were selected to obtain representative images. Protein expression levels were standardized to those of GAPDH in different samples. 

Immunofluorescence and Confocal Microscopy

Briefly, scleral fibroblasts grown on coverslips were fixed in 4% paraformaldehyde (PFA) for 30 minutes, permeabilized with 0.3% Triton X-100, and blocked with 3% BSA. Then, the cells were incubated overnight at 4°C with the corresponding primary antibodies against YAP (1:100; Cell Signaling Technology), integrin α1 (1:100, Abcam), and vinculin (1:100; Cell Signaling Technology). After extensive washing with PBS, the cells were protected from light and subjected to immunofluorescence staining, which included incubation with FITC-labeled secondary antibodies (1:100, Jackson ImmunoResearch Labs), for two hours at room temperature. DAPI was used to stain the nuclei (Invitrogen), and Alexa-conjugated phalloidin (Cytoskeleton Inc., Denver, CO, USA) was used to visualize actin. Immunofluorescence images were captured using a Leica SP8 confocal microscope (Leica, Wetzlar, Germany). In the quantification step, the fluorescence intensity was measured via ImageJ. The total fluorescence intensity for a designated region of interest was calculated and then normalized to the area of that region of interest. The final calculation is expressed using the following formula: Mean fluorescence intensity = Total integrated density (IntDen)/Area. 

Statistical Analysis

GraphPad Prism Version 8.0 was used for statistical analysis. The data are expressed as the mean ± standard deviation (SD). Student's paired t test was used to analyze the differences between FD eyes and control eyes. Independent t tests or one-way ANOVA were used to compare measurements between different guinea pigs and different groups of cells. Then, post hoc multiple comparisons (LSD for data with equal variance) were performed to determine pairwise differences. The Mann-Whitney U test was used to compare two quantitative variables in humans with uneven variance. A P value < 0.05 was considered statistically significant. 

Results

YAP Expression Levels are Decreased in the Sclera of Guinea Pigs and Humans With Myopia

In our previous studies, we observed a significant development of myopia in guinea pigs after four weeks of FD (Supplementary Fig. S1). Subsequent proteomics analysis revealed notable alterations in the pathways related to scleral remodeling.¹⁶ The functions of cellular movement and cellular assembly and organization (CMCSO) exhibited the most pronounced changes, with approximately one third of the proteins being altered as a result of myopia. To further investigate the connection between CMCSO and myopia, we conducted an ingenuity pathway analysis (IPA)—a prediction analysis using core analysis and upstream regulator prediction analysis (Fig. 1A). This approach allowed us to identify upstream regulators and make predictions regarding their activation or inhibition on the basis of the observed gene expression changes within the CMCSO in our experimental dataset. Remarkably, the findings pointed toward YAP, a transcription coregulator, as a pivotal player upstream of the CMCSO that is capable of exerting a substantial impact on the progression of myopia. 

Figure 1.

 

YAP expression is decreased in the sclera of guinea pigs and humans with myopia. (A) Protein function and network prediction analysis using ingenuity pathway analysis (IPA). (B, C) WB analysis of YAP and COL1A1 in the scleral tissues of guinea pig FD and control eyes (n = 5). (D, E) WB analysis of YAP and COL1A1 in the scleral tissues of human myopic eyes (n = 4). (G) ELISA analysis of the serum levels of the YAP-targeted gene CTGF in patients with mild myopia and high myopia. (F, H) Spearman correlation analysis between the serum CTGF and diopter and AXL. The data are expressed as the mean ± SD (*P < 0.05, **P < 0.01).

To validate this outcome, we conducted a WB analysis to compare the levels of YAP expression in the sclera of guinea pigs with and without myopia (Figs. 1B, 1C). The data revealed significantly lower YAP expression in FD eyes than in fellow eyes (Figs. 1B, 1C). Moreover, the expression of COL1A1, a marker associated with myopia remodeling, was also diminished in the myopia group (Figs. 1B, 1C). Importantly, these findings were corroborated by human studies (Figs. 1D–H). The human demographic and basic ocular parameters are presented in Supplementary Tables S1 through S3. YAP expression was strikingly decreased in human myopic sclera (Figs. 1D, 1E), paralleled by a reduction in the expression of CTGF, a prominent downstream target of YAP, in human myopic serum (Fig. 1G). Additionally, the level of CTGF was positively correlated with the diopter and negatively correlated with the eye axis (Figs. 1F, 1H). 

The YAP Expression Level is Decreased, and its Subcellular Location is Altered in the Scleral Fibroblasts Grown on Soft Substrates

To gain further insight into the molecular mechanisms underlying the role of YAP in myopia, we cultured scleral fibroblasts on both stiff and soft substrates (Fig. 2A). The choice of a soft substrate was based on previous research indicating its ability to replicate the microenvironment associated with myopia.¹⁷ By culturing the scleral fibroblasts on a soft substrate, we aimed to recreate the physical and pathological changes in scleral cells observed in myopia. Notably, our experimental results demonstrated a significant decrease in the expression of YAP in the scleral fibroblasts cultivated on a soft substrate (Figs. 2B, 2C). Furthermore, we observed a concurrent reduction in the levels of Ankrd and CTGF, two downstream target genes of YAP (Fig. 2D). Additionally, the results obtained from immunostaining of YAP revealed not only a reduction in the expression of YAP in the scleral fibroblasts on the soft substrate but also a notable alteration in its subcellular location (Figs. 2E, 2F). 

Figure 2.

 

Matrix stiffness mediated YAP expression and nuclear localization in scleral fibroblasts. (A) Illustration of in vitro experiments involving scleral fibroblasts cultured on stiff and soft substrates. (B, C) WB analysis of YAP expression in scleral fibroblasts cultured on stiff and soft substrates (n = 3). (D) qRT‒PCR analysis of the mRNA expression of the YAP target genes Ankrd and CTGF in scleral fibroblasts cultured on stiff and soft substrates (n = 3). (E) Representative immunofluorescence staining of YAP in scleral fibroblasts cultured on stiff and soft substrates. (F) Subcellular distribution of YAP represented by decreased relative nuclear YAP intensities (n = 12, nine points in stiff and soft substrates respectively, adopted from three independent samples for each group). (G, H) WB analysis of COL1A1 levels in scleral fibroblasts cultured on stiff and soft substrates or treated with the YAP inhibitor verteporfin (n = 3). The data are expressed as the mean ± SD (*P < 0.05, **P < 0.01).

In the myopia-like environment, there was an increased presence of the YAP protein within the cytoplasm as opposed to the nucleus. Specifically, nuclear YAP accounted for approximately 76% of the total YAP in the scleral fibroblasts on the stiff substrate, whereas this percentage decreased to approximately 40% on the soft substrate (Fig. 2F). Moreover, the expression of COL1A1 was diminished in both the scleral fibroblasts on the soft substrate and the scleral fibroblasts treated with VP, an inhibitor of YAP (Figs. 2G, 2H). Therefore the changes observed in the downstream targets of YAP in the scleral fibroblasts on the soft substrate are likely due to both reduced YAP expression and a lower accumulation of nuclear YAP. 

Integrin α1β1 Regulates the Subcellular Localization of YAP

Previous studies have shown that the nuclear localization of YAP in basal layer skin keratinocytes may be stimulated by integrin–Src and/or PI3K signaling.¹⁸ Our proteomics results revealed significant disturbances in the integrin pathway in myopic sclera. On the basis of these findings, we hypothesized that the increased cytoplasmic presence of the YAP protein identified in the scleral fibroblasts on the soft substrate could be attributed to disrupted expression of integrin proteins. To address this hypothesis, we initially examined the expression of Itgα1, α2, α10, α11, and β1 by qPCR, as these proteins are the primary collagen-binding receptors. Our findings revealed a significant decrease in the mRNA levels of Itgα1 and β1 in the sclera of guinea pigs with myopia (Fig. 3A). Furthermore, the WB results confirmed this observation, showing a significant reduction in the protein levels of Itgα1 and β1 in the sclera of myopic guinea pigs (Figs. 3B, 3C). 

Figure 3.

 

Matrix stiffness mediates YAP activity and YAP nucleocytoplasmic shuttling via integrin α1β. (A) qRT‒PCR analysis of the mRNA expression of Itgα1, Itgα2, Itgα10, Itgα11, and Itgβ1 in guinea pig FD sclera (n = 5). (B, C) WB analysis of Itgα1 and Itgβ1 in guinea pig FD sclera (n = 5). (D–F) WB analysis of Itgα1 and Itgβ1 in scleral fibroblasts cultured on stiff and soft substrates (n = 3). (G) qRT‒PCR analysis of the mRNA expression of the YAP target genes CTGF and Ankrd in scleral fibroblasts cultured on stiff and soft substrates treated with siItgα1 (n = 3). (H, I) WB analysis to validate the effectiveness of siItgα1. (J, K) WB analysis of YAP expression in scleral fibroblasts cultured on stiff and soft substrates treated with siItgα1 (n = 3). (L, M) Representative immunofluorescence staining of YAP in scleral fibroblasts cultured on stiff and soft substrates treated with siItgα1. The data are expressed as the mean ± SD (*P < 0.05, **P < 0.01).

In an in vitro model, we also noted a dramatic decrease in the protein levels of Itgα1 and β1 in the scleral fibroblasts cultured on the soft substrate (Figs. 3D–F). Further investigation revealed that while siRNA targeting Itgα1 did not alter the expression of YAP on either the stiff or soft substrate, it significantly decreased the expression of Ankrd and CTGF, specifically on the stiff substrate (Figs. 3G–K). The subsequent immunofluorescent results suggested that this substantial reduction in downstream genes may be attributed to decreased levels of YAP in the nucleus (Figs. 3L, 3M). Overall, our findings indicate that disruption of integrin proteins in myopic sclera may hinder the accumulation of YAP in the nucleus and subsequently reduce the expression of its downstream targets, ultimately contributing to the modulation of elasticity in the eyes. 

Integrin α1β1 Triggers YAP Accumulation in the Nucleus via Actin Polymerization

How does integrin α1β1 affect the subcellular localization of YAP? To address this inquiry, we investigated the effects of jasplakinolide (an F-actin stabilizer) and CytoD (an F-actin polymerization inhibitor) on the scleral fibroblasts cultured on both stiff and soft substrates. Our findings revealed that the administration of jasplakinolide or CytoD did not alter the expression levels of YAP on either stiff or soft substrates (Figs. 4A–D). However, jasplakinolide treatment significantly increased the expression of CTGF and Ankrd in the scleral fibroblasts cultured on soft substrate, whereas CytoD treatment decreased the expression of CTGF and Ankrd in scleral fibroblasts cultured on stiff substrates (Fig. 4E). Moreover, the results of the immunofluorescent analysis demonstrated that in conjunction with integrin α1 siRNA application, the administration of jasplakinolide promoted YAP accumulation in the nucleus (Figs. 4F, 4G). Therefore we suggest that integrin α1 triggers YAP accumulation in the nucleus and enhances the expression of downstream targets via actin polymerization. 

Figure 4.

 

YAP activity and nuclear localization are modulated by actin polymerization. (A, B) WB analysis of YAP expression in scleral fibroblasts cultured on stiff and soft substrates treated with cytochalasin D (CytoD) (n = 6). (C, D) WB analysis of YAP expression in scleral fibroblasts cultured on stiff and soft substrates treated with jasplakinolide (n = 3). (E) qRT‒PCR analysis of the mRNA expression of the YAP target genes CTGF and Ankrd in scleral fibroblasts cultured on stiff and soft substrates treated with CytoD or jasplakinolide (n = 3). (F, G) Immunostaining and proportion of nuclear YAP in scleral fibroblasts treated with siItgα1 and siItgα1 + jasplakinolide. The data are expressed as the mean ± SD (*P < 0.05, **P < 0.01).

The Integrin α1β1–Dependent YAP Pathway Regulates the Expression of COL1A1

We previously demonstrated that YAP plays a role in regulating the expression of COL1A1. Here, we provide further evidence supporting this notion. Specifically, silencing YAP by siRNA (Figs. 5A, 5B) interference in scleral fibroblasts cultured on stiff substrate significantly decreased COL1A1 expression (Figs. 5E, 5F). Conversely, overexpressing YAP (Figs. 5C, 5D) in scleral fibroblasts cultured on soft substrates increased COL1A1 expression (Figs. 5G, 5H). Furthermore, the application of integrin α1 siRNA to scleral fibroblasts cultured on the stiff substrate decreased the expression of COL1A1 (Figs. 5I, 5J), whereas the administration of jasplakinolide promoted the expression of COL1A1 in scleral fibroblasts cultured on the soft substrate (Figs. 5K, 5L). These findings strongly suggest that the expression of COL1A1 is regulated through the integrin α1–dependent YAP pathway. 

Figure 5.

 

The integrin-YAP pathway targets COL1A1 expression in scleral fibroblasts subjected to matrix stiffness stimuli. (A, B) WB analysis of YAP expression in scleral fibroblasts treated with siYAP (n = 3). (C, D) WB analysis of YAP expression in scleral fibroblasts treated with YAP plasmid (n = 3). (E, F) WB analysis of COL1A1 expression in scleral fibroblasts cultured on stiff substrates treated with siYAP (n = 3). (G, H) WB analysis of COL1A1 expression in scleral fibroblasts cultured on soft substrates treated with YAP plasmid (n = 3). (I, J) WB analysis of COL1A1 expression in scleral fibroblasts cultured on sitff substrates treated with siItgα1. (K, L) WB analysis of COL1A1 expression in scleral fibroblasts cultured on soft substrates treated with jasplakinolide (n = 3). The data are expressed as the mean ± SD (*P < 0.05, **P < 0.01).

In Vivo Targeting of YAP Expression and Activity Modulated FD Myopia Development and COL1A1 Expression

FD guinea pigs were used as an in vivo intervention model. FD eyes were administered subconjunctival injections of verteporfin (700 nM) to inhibit YAP, along with xmu-mp-1 (10 µM) and jasplakinolide (25 nM) to activate YAP. Additionally, another group of guinea pigs solely underwent FD as a control (Fig. 6A). After four weeks, AXL and refraction were measured. Inspiringly, we observed an attenuation of myopia development (calculated as the difference between the FD eye and the control eye) in the xmu-mp-1 group compared with the control group (Figs. 6B, 6C). Concurrently, interocular differences in refraction were significantly greater in the VP group than in the control group, and differences in AXL also increased (Figs. 6B, 6C). Similarly, the elongation of AXL and changes in the diopters of FD eyes in response to jasplakinolide were also significantly inhibited (Figs. 6B, 6C). Collectively, these results support the crucial role of YAP signaling in the progression of myopia. 

Figure 6.

 

YAP overexpression retarded myopia development under FD. (A) An illustration of the in vivo intervention in guinea pigs. (B, C) Interocular differences in refraction and AXL were measured in the FD group (n = 16), FD group with jasplakinolide (n = 6), FD group with VP (n = 6), and FD group with xmu-mp-1 (n = 7). (D–G) WB analysis of COL1A1 in the FD groups with verteporfin, xmu-mp-1 or jasplakinolide (n = 3). The data are expressed as the mean ± SD (*P < 0.05, **P < 0.01).

Furthermore, scleral levels of COL1A1 were assessed in this study. In the control group, the levels of COL1A1 were lower in FD eyes than in fellow eyes. However, this decrease was effectively suppressed in FD eyes treated with xmu-mp-1 (Figs. 6D, 6G) and jasplakinolide (Figs. 6E, 6G). Moreover, a significant decrease in COL1A1 was observed in FD eyes in the VP group (Figs. 6F, 6G). These results provide evidence that targeting YAP activity can effectively modulate COL1A1 expression. 

Discussion

Scleral ECM remodeling is widely recognized as the primary factor contributing to the development of myopia. However, the specific regulatory mechanisms involved in this process remain elusive. In this study, we aimed to investigate the mechanical regulation of scleral fibroblasts by ECM stiffness and its crucial role in scleral ECM remodeling and myopia development. Our findings revealed a previously unreported role of YAP nuclear localization and activity in myopic scleral remodeling. Notably, we observed that a softer matrix inhibited the expression of integrin α1β1, leading to suppressed polymerization of F-actin in scleral fibroblasts. As a result, the nuclear translocation of YAP was reduced, ultimately leading to decreased expression of COL1A1. Through the manipulation of integrin α1β1–F-actin–YAP signaling both in vitro and in vivo, we were able to confirm the significance of mechanotransduction in scleral remodeling during myopia (Fig. 7). 

Figure 7.

 

Schematic model illustrating the underlying mechanisms by which matrix stiffness regulates YAP activity and the nuclear localization of scleral fibroblasts during scleral ECM remodeling in myopia.

YAP, a key mediator of the Hippo pathway, has been shown to potentially be associated with myopia at the gene expression level in both marmosets and mouse experimental myopic models through RNA-seq.^19,20 Considering the growing significance of YAP in connecting mechanical cues and the Hippo pathway, we questioned whether a mechanosensitive molecular mechanism could lead to YAP dysregulation in myopia. Initially, we analyzed human scleral tissues and found significantly lower YAP expression in myopic eyes compared to fellow eyes. However, differences in age among the donors (Supplementary Table S2) may have influenced these results. To strengthen the validity of these preliminary findings, we conducted further validation using in vivo myopic models. Our in vivo animal experiments revealed that the expression and activity of YAP in scleral tissue were significantly downregulated in the FD eyes of guinea pigs. Additionally, the serum level of CTGF, a downstream target of YAP, is lower in highly myopic patients than in those with mild myopia, further strengthening the association between YAP dysregulation and myopia. Previous studies have demonstrated a substantial decrease in scleral stiffness during myopia development.^15,21–23 In myopic guinea pigs, for everyone more diopter induced, the bulk modulus in the vertical section would decrease by 0.036 GPa.¹⁷ Therefore our in vitro study aimed to investigate the response of scleral fibroblasts to different stiffness matrices. It was found that a soft matrix inhibited the expression and activity of YAP in scleral fibroblasts, as evidenced by increased cytoplasmic retention and reduced downstream target expression. These changes also led to a decrease in COL1A1 expression. 

Interestingly, when YAP activity was activated via a plasmid or an agonist (jasplakinolide), scleral fibroblasts presented increased COL1A1 expression even when cultured on a soft matrix, suggesting that activating YAP can overcome the physical constraints of COL1A1 expression on soft substrates. Recent studies have highlighted the role of YAP in extracellular matrix (ECM) remodeling and collagen production in various fibrotic diseases.^11,24–27 Futakuchi et al.²⁸ demonstrated that YAP promotes TGFβ1-mediated fibrosis and myofibroblast differentiation during conjunctival fibrosis. Additionally, an in vitro stretching experiment with scleral fibroblasts revealed an increase in type 1 collagen protein levels.²⁹ Taken together, these findings suggest that scleral fibroblasts can respond to the mechanical properties of their extracellular environment through YAP-mediated mechanotransduction, which in turn affects collagen expression. Importantly, our results provide a novel molecular link between ECM stiffness and the progression of myopia. We propose that a weakened biomechanical environment in the sclera can initiate a vicious cycle of scleral remodeling and accelerate myopia progression through positive feedback. 

Furthermore, our study focused on investigating the mechanism by which YAP dysregulates scleral fibroblasts. Integrin, a mechanosensor that transmits force to the actin cytoskeleton, plays a crucial role in this process. Different subtypes of integrins respond to various stimuli, and this response is specific to distinct tissues. For example, integrin β3 is responsible for mediating the response to unidirectional shear stress in aortic endothelial cells and focal force stimulation in osteocytes.^30,31 Silencing integrin α1 through siRNA enhanced the cytoplasmic retention of YAP and reduced the expression of YAP target genes, while promoting F-actin polymerization counteracted this effect. Therefore our results indicate that matrix stiffness can regulate the translocation of YAP to the nucleus through integrin α1β1-mediated F-actin polymerization in scleral fibroblasts. 

Acknowledgments

Supported by the National Natural Science Foundation of China [grant numbers 82371092, 81770953], National Key Research and Development Program of China [grant number 2024YFC2510802], and Shang-hai Jiao Tong University Medical Engineering Cross Research [grant number YG2021ZD18]. 

Disclosure: X. Liu, None; Y. Yuan, None; Y. Wu, None; C. Zhu, None; Y. Liu, None; B. Ke, None