Distinct Motility and Pro-fibrotic effect of Renal Tubule-derived Extracellular Vesicles in Fibrotic Extracellular Matrix.

Abstract

Extracellular vesicles (EVs) facilitate intercellular communication by traversing the extracellular matrix (ECM). However, their motility within fibrotic ECM and its role in fibrosis development remain unclear. We engineered stress-relaxing (SR) hydrogels of tunable stiffness (2, 50 kPa) through dynamic crosslinking of short peptide (WGG(KA)) and heparin to mimic normal and fibrotic ECM. Super-resolution nanoimaging and quantitative three dimensional (3D) single-particle tracking (SPT) of single EV were performed, and the motion dynamics was quantified. The interplay between EVs and ECM was further investigated, particularly its effects on fibroblast activation and renal fibrosis. It was identified that both normal and fibrotic kidney-derived EVs exhibited confined Brownian-like motion according to 3D SPT, with enhanced mobility in the stiffer (50 kPa) hydrogel. Notably, fibrotic tubule-derived EVs carried higher levels of integrin β6 (ITGB6), which reduced their mobility within the hydrogel-based ECM mimic, as confirmed by the restoration of motility upon ITGB6 blocking or digestion. This suggested that EV motility may be influenced by the interplay between ECM stiffness and the intrinsic properties of the EVs. Furthermore, enrichment of ITGB6 on fibrotic tubule-derived EVs promotes local retention, thereby increasing EV-fibroblast interaction and profibrotic signaling. This indicated the underappreciated role of EV in fibrosis related to its motility and its interplay with fibroblast in fibrotic niche. Our study provides new insights into the mechano-dependent mechanisms governing EV motility within the fibrotic ECM. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) play key roles in cell communication, but how they move through fibrotic tissue remains poorly understood. This study reveals that kidney-derived EVs exhibit confined Brownian-like motion within engineered hydrogels mimicking fibrotic extracellular matrix. We found that EVs from diseased kidneys carry elevated integrin β6, which restricts their mobility and promotes fibroblast activation via LAP-TGF-β1 signaling. This work establishes a mechano-dependent paradigm linking matrix stiffness and EV motility to fibrosis progression, offering new insights into how EVs navigate dense tissue environments. The findings advance our understanding of fibrotic disease mechanisms and highlight EVs as potential therapeutic targets or delivery vehicles in stiffened tissues.