Piezo1 Promotes Scleral ECM Remodeling via Mechanical Strain–Induced Fibroblast Differentiation

Summary

This study combines a rat ocular hypertension model with in vitro mechanical strain, agonist/inhibitor, and siRNA experiments in human scleral fibroblasts to show that Piezo1 expression is elevated during scleral extracellular matrix remodeling and is functionally involved in myofibroblast differentiation.

Abstract

Mechanical strain-induced myofibroblast differentiation is a pivotal mechanism underlying scleral extracellular matrix (ECM) remodeling in glaucoma. However, the conversion of mechanical strain into biochemical signals within scleral fibroblasts remains inadequately elucidated. This study sought to investigate the role of Piezo1 in cellular proliferation and differentiation, in the context of mechanical strain-induced myofibroblast differentiation during scleral ECM remodeling. The study design combines in vivo expression observations from a one‑week rat ocular hypertension model with in vitro mechanistic assays using cyclic mechanical stretch (10% at 0.5 Hz for up to 24 h) and pharmacological modulation of Piezo1 (agonist Yoda1, inhibitor GsMTx4, and siRNA knockdown) in primary human scleral fibroblasts (HSF cells). Our findings demonstrate that Piezo1 is expressed in scleral tissue and HSF cells. In the in vivo arm, a significant increase in Piezo1 protein levels was observed in the sclera of ocular hypertensive rats after one week, which coincided with elevated expression of type I collagen (COL1) and α‑smooth muscle actin (α‑SMA). In the in vitro experiments, mechanical stretch (8–24 h) upregulated Piezo1 expression in HSF cells. Moreover, activation of Piezo1 with Yoda1 (24 h) facilitated HSF cell proliferation and enhanced the expression of α‑SMA and COL1. Conversely, knockdown or inhibition of Piezo1 suppressed COL1 expression and hindered myofibroblast differentiation under mechanical strain. Additionally, Piezo1 activation led to an upregulation of Yes‑associated protein (YAP) expression, whereas mechanical strain‑induced YAP expression was diminished upon Piezo1 knockdown or inhibition. In summary, our findings suggest that Piezo1 plays a role in mechanical strain‑induced expression of key ECM components in the sclera in the context of glaucoma and may represent a potential therapeutic target requiring further in vivo validation.

Introduction

Glaucoma is a leading cause of irreversible blindness characterized by progressive degeneration of retinal ganglion cells (RGCs) and optic nerve head (ONH) axons, affecting 76 million people worldwide in 2020^1,2. Elevated intraocular pressure (IOP) is the greatest risk factor for glaucoma³ and IOP reduction is currently the only available option to delay glaucoma development and progression⁴. The ONH, the structure where the RGC axons exit the eye, is the principal site of damage in glaucoma. IOP elevation induces mechanical strain and displacement in the ONH, resulting in degeneration of the RGC axons within the laminar cribrosa (LC), eventually leading to glaucomatous optic neuropathy^5,6.

The peripapillary sclera (PPS) is the principal load-bearing tissue of the ONH, with the mechanical properties of PPS determining the ONH response to IOP-induced mechanical strain^7,8. Finite element modeling and ex vivo studies confirmed that the LC biomechanical strain relies on scleral stiffness, and enhanced PPS stiffness contributes to reduced strain in the LC^9,10. Then, the variation in stiffness^11,12 and thickness¹³ of PPS can alter the IOP-related biomechanical properties of the ONH, further changing the susceptibility to glaucomatous optic nerve damage^14,15. In biomechanical studies, the ocular rigidity of glaucomatous sclera was greater than normal sclera in postmortem human eyes ^16,17, and peripapillary scleral stiffening has been consistently observed in both human glaucoma and experimental animal models^18,19,20. These data suggest that stiffening of the sclera may be a protective response through decreased IOP-induced ONH deformation during glaucoma.

The biomechanical characteristics of the sclera tissue are a result of the composition of the extracellular matrix synthesized by fibroblasts²¹. Recent evidence suggests that mechanical strain arising from increased IOP is capable of activating scleral fibroblasts residing within the sclera²², promote myofibroblast differentiation²³ and result in ECM remodeling^24,25. The mechanical strain-induced myofibroblast transformation is a key process during scleral ECM remodeling in glaucoma, but it is unclear how the mechanical strain is converted into biochemical signals and cellular responses in scleral fibroblasts.

Piezo1 is one member of a novel distinct nonselective cationic mechanosensitive channel family involved in fibrosis of several organs, including cardiac and lung skin^26,27. Mechanical strain can activate the cellular Piezo1 channels leading to increased Ca²⁺ influx²⁸, thereby calcium-dependent signaling to regulate a variety of cellular activities, including the proliferation, motility, and differentiation of fibroblasts^29,30. Recent studies revealed that under mechanical strain, Piezo1 promotes skin fibrotic processes by stimulating fibroblast proliferation, motility, and differentiation³¹. This study includes two complementary components: a descriptive in vivo study of Piezo1 expression in a rat ocular hypertension model, and a mechanistic in vitro study using primary human scleral fibroblasts under cyclic mechanical strain. We hypothesized that mechanical strain activates Piezo1, which promotes myofibroblast differentiation and ECM component expression in scleral fibroblasts, potentially via YAP signaling.

Protocol

Male Sprague‑Dawley rats (8 weeks of age) were sourced from the Fudan University Children's Hospital Experimental Animal Center. All rats were ad libitum and housed under SPF conditions at 22±1 ℃ and a 12 h day/night cycle. All animal experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Review Board and Ethics Committee of Children's Hospital of Fudan University (2022JS-EKYY-019). Human scleral tissues used to derive primary human scleral fibroblasts were obtained from donor sclera in accordance with institutional guidelines, and all related procedures were approved by the Institutional Review Board of Fudan University (2024-YS-187).

Experimental design

The present study employed both in vivo and in vitro approaches, conceptually connected by mechanical strain, to examine the contribution of Piezo1 to scleral ECM remodeling. In vivo, we used a rat ocular hypertension model to examine Piezo1 expression under elevated IOP. In vitro, we subjected primary human scleral fibroblasts to cyclic mechanical stretch to model the mechanical strain experienced by the sclera in glaucoma, and we manipulated Piezo1 activity using pharmacological agonist (Yoda1), inhibitor (GsMTx4), and siRNA knockdown.

Experimental chronic ocular hypertension (OHT) model

A total of 18 male SD rats were used. In each rat, the right eye received carbomer injection to induce ocular hypertension (OHT), with the contralateral left eye serving as the control group (NT). Three independent biological replicates of the whole experiment were performed, giving a total of 18 eyes per group. The experimental unit was the individual eye. Investigators performing IOP measurements and Western blot analysis were blinded to group allocation.

An experimental chronic OHT model was generated in rats as previously described²⁵. Briefly, rats were anesthetized with 10% chloral hydrate, and after pupillary dilation by tropicamide, carbomer solution (0.3%, 20 µL) was injected into the anterior ocular chamber. Chlortetracycline hydrochloride was applied to prevent infection. IOP measurements were performed with a rebound tonometer after 3 and 7 days. Tissues from the sclera were collected one week after the model was considered established.

The scleral tissues were isolated as described previously in detail²⁵. Following anesthesia, the rats were perfused transcardially with 4% formaldehyde in PBS. Extraneous orbital tissue was dissected from the globe, and the anterior segment structures (cornea, iris, lens) and the vitreous were carefully removed. The retina and choroid were gently scraped from the inner scleral wall. The posterior sclera tissues were collected and identified by pathological H&E staining.

Cell cultivation

Primary human scleral fibroblasts (HSF cells) were a previously established cell line from human donor sclera³². Three independent biological replicates (cells from three different donors) were used for each experiment. HSF cells were maintained in DMEM supplemented with 15% fetal bovine serum and 1% penicillin‑streptomycin under standard culture conditions (37 °C, 5% CO2, humidified atmosphere). Cultures were split every four days, and HSF cells between passages 5 and 9 were employed for all experiments. HSF cells were confirmed by immunofluorescence using collagen type I and α-smooth muscle actin cell markers.

Application of mechanical stimulation

The cyclic mechanical stretch system was used to produce mechanical cyclic stretch. A seeding density of 2×10⁵ cells/mL was used for HSF cells plated in six‑well plates with collagen‑coated flexible bottoms. Stretch group cells were exposed to cyclic stretch at 10% amplitude and 0.5 Hz as described in our previous study³². Cells cultured in stretch plates, but left static served as controls. Piezo1 siRNA knockdown experiment: Cells received a 48‑h transfection of Piezo1 siRNA or negative control siRNA prior to mechanical stretch. After transfection, the cells underwent cyclic mechanical stretching for 24 h. Four groups were included: (A) control siRNA‑transfected cells without stretch; (B) control siRNA‑transfected cells with stretch; (C) Piezo1 siRNA‑transfected cells without stretch; (D) Piezo1 siRNA‑transfected cells with stretch. Piezo1 inhibitor (GsMTx4) experiment: Cells were treated with 5 µM GsMTx4 prior to and during mechanical stretch. Four groups were included: (A) untreated cells without stretch; (B) untreated cells with stretch; (C) GsMTx4‑treated cells without stretch; (D) GsMTx4‑treated cells with stretch (with GsMTx4 present throughout). Piezo1 agonist (Yoda1) experiment: Cells were cultured in the presence of 5 µM Yoda1 for 24 h. Two groups were included: (A) untreated (vehicle) control; (B) Yoda1‑treated cells.

Western blotting

Protein levels were assessed by Western blotting following routine procedures. Briefly, we lysed scleral or cultured fibroblast samples in RIPA buffer. Protein (20 µg) was resolved by SDS‑PAGE and subsequently blotted onto nitrocellulose membranes. The membranes were then blocked with 5% BSA for 1 h at ambient temperature, followed by overnight incubation at 4 °C with the appropriate primary antibodies. The antibodies used included Piezo1, collagen type I, α-smooth muscle actin, YAP and GAPDH. The Piezo1 antibody has been validated by the manufacturer for Western blot and immunofluorescence in rat and human tissues, with knockout/knockdown data available on the product sheet. Following three washes with TBST, we incubated the membranes with appropriate HRP‑conjugated secondary antibodies (anti‑rabbit or anti‑mouse) for 1 h at room temperature. Band intensities were then measured using image analysis software, and each target band’s signal was normalized to the GAPDH signal from the same lane. The resulting data are presented as fold‑change relative to the control condition.

Cell proliferation

Cell proliferation was assessed by a colorimetric viability assay following the supplied protocol. We added a tetrazolium compound (10 µL/mL) to each well and incubated the plates for 2 h under standard culture conditions (37 °C, 5% CO2). Absorbance was then read at 450 nm.

Small interfering RNA transfection

Piezo1 siRNA transfection was performed as previously described³¹. The sequences were as follows: Piezo1-siRNA, 5’-AGAAGAAGAUCGUCAAGUATT-3' (sense) and 5’-UACUUGACGAUCUUCUUCUTT-3' (antisense), negative control (NC) siRNA, 5’-GUGAGCGUCUAUAUACCAUTT-3' (sense) and 5’-AUGGUAUAUAGACGCUCACTT-3' (antisense).

Immunofluorescence

For immunofluorescence, cultured cells were fixed with 4% paraformaldehyde (20 min, room temperature), permeabilized with 0.1% Triton X‑100 (40 min), and then blocked with 10% BSA. Tissue sections underwent overnight fixation in 4% paraformaldehyde at room temperature, followed by permeabilization and blocking. Specimens were incubated with primary antibodies against Piezo1 and α‑SMA. Subsequently, we applied fluorescent dye‑conjugated secondary antibodies: green‑labeled anti‑mouse (for α‑SMA) and red‑labeled anti‑rabbit (for Piezo1), each at 1:500 dilution. Nuclei were counterstained with DAPI, and images were captured with a fluorescence microscope.

Agonist and inhibitor treatment

HSF cells were treated with 5 µM GsMTx4 and 5 µM of the Piezo1 agonist Yoda1 based on previous studies³³.

Statistical analysis

Each experiment was carried out in triplicate. Results are presented as mean ± standard deviation (SD). For comparisons between two groups, we applied a two‑tailed Student’s t‑test. Multiple group comparisons were performed with one‑way ANOVA followed by Tukey’s post‑hoc test. The normality of data was checked by the Shapiro‑Wilk test, and homogeneity of variances was evaluated using Levene’s test. Statistical significance was defined as p < 0.05. All statistical analyses were conducted with dedicated statistical software.

Results

Piezo1 is expressed in rat sclera tissues and HSF cells

First, we examined Piezo1 expression in both rat scleral tissues (in vivo) and cultured human scleral fibroblasts (in vitro). To interrogate tissue‑specific expression of Piezo1, the BioGPS gene annotation database was used³⁴. BioGPS analysis revealed that Piezo1 is highly expressed in various eye tissues, including retinal pigment epithelium, iris, cornea, and eyecup (Figure 1...

Discussion

Elevated intraocular pressure (IOP) induces mechanical strain in the optic nerve head and laminar cribrosa, contributing to retinal ganglion cell degeneration^38,39. The peripapillary sclera (PPS) is a major load‑bearing tissue that influences ONH biomechanics⁴⁰. Previous studies have shown that mechanical strain induces scleral fibroblast‑to‑myofibroblast differentiation and extracellular matrix (ECM) remodeling, leading ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.3% carbomer solution (Carbomer 940 polymer)	Solarbio, Shanghai, China	—	20 μL
10% chloral hydrate	Macklin, Shanghai, China	—	3 mL/kg
Alexa Fluor 488-conjugated goat anti-mouse secondary antibody	Thermo Fisher Scientific, Waltham, MA, USA	—
Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody	Thermo Fisher Scientific, Waltham, MA, USA	—
BioFlex six-well culture plates (coated with collagen I)	Flexcell Int Corp, Hillsborough, NC, USA	—
Chlortetracycline hydrochloride	Zhenhua Pharmaceutical, Yunnan, China	—
COL1 primary antibody	Novus Biologicals, CO, USA	—
DMEM	Gibco, Grand Island, NY, USA	—
FBS	Gibco Life Technologies, Australia	—
GAPDH primary antibody	Bioworld, MN, USA	—
GraphPad Prism version 8.4	Inc., San Diego, CA, USA	—
Nikon Eclipse E800 microscope	Nikon, Melville, NY, USA	—
Nitrocellulose membranes	Pierce, Rockford, IL, USA	—
Penicillin-streptomycin	Hyclone, South Logan, UT, USA	—
Piezo1 primary antibody	Novus Biologicals, CO, USA	—
Rebound tonometer (TonoLab)	Icare, Espoo, Finland	—
RIPA buffer	Thermo Fisher Scientific, Shanghai, China	—
The Flexcell FX-5000 Tension System	Flexcell International Corporation	—	Used to apply mechanical cyclic stretch to HSF cells.
Tropicamide	Bausch Lomb Freda, Shandong, China	—
YAP primary antibody	Cell Signaling Technology, Danvers, USA	—
α-SMA primary antibody	Abcam, Cambridge, UK	—