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, re....

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 ap....

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	—