Matrix Stiffness Controls Lipid Homeostasis in Human Corneal Endothelial Cells via a Piezo1-PI3K/AKT-mTOR-PPARγ Mechanotransduction Axis

Substrate stiffness is a critical mechanical cue that regulates cellular behaviors, yet its role in governing lipid homeostasis of human corneal endothelial cells (HCECs) remains poorly understood. Here, we developed a polyacrylamide hydrogel platform with tunable stiffnesses (25, 50, and 100 kPa) covering the pathophysiological range of Descemet's membrane to investigate stiffness-dependent lipid metabolic reprogramming in HCECs. All hydrogels exhibited excellent transparency (>95%) and good biocompatibility. We found that soft substrates (25 kPa) promoted substantial accumulation of neutral lipids and free cholesterol in HCECs. Mechanistically, soft substrates upregulated SREBP1 and its target genes to enhance fatty acid synthesis while suppressing CPT1-mediated fatty acid oxidation, and simultaneously upregulated LDLR and SREBP2 to promote cholesterol uptake and synthesis while downregulating the LXRα-ABCA1/ABCG1 axis to impair cholesterol efflux. Notably, PPARγ emerged as a key regulator of stiffness-dependent lipid homeostasis; its expression was positively correlated with substrate stiffness, and pharmacological activation of PPARγ reversed soft substrate-induced lipid accumulation. Furthermore, the Piezo1-PI3K/AKT-mTOR signaling axis was identified as an upstream mechanotransduction pathway mediating stiffness-regulated PPARγ expression. Analysis of transcriptomic datasets from Fuchs endothelial corneal dystrophy (FECD) patients revealed dysregulation of lipid metabolism genes consistent with our findings. Together, these results demonstrate that substrate stiffness governs HCEC lipid homeostasis through a Piezo1-PI3K/AKT-mTOR-PPARγ mechanotransduction cascade, and that matrix softening promotes abnormal lipid accumulation by reprogramming fatty acid and cholesterol metabolic networks. Our findings establish a mechanistic link between the mechanical microenvironment and lipid metabolism in corneal endothelial cells, providing insights into FECD pathogenesis and a theoretical framework for designing stiffness-tuned biomaterials for corneal endothelial regeneration.