Piezo1 activation mediates stiffness-induced aortic medial calcification: Pharmacological evidence from agonist and antagonist studies.

Vascular calcification (VC) is a hallmark of cardiovascular disease progression, with matrix stiffness-induced VC playing a critical role. However, the mechanical transmission mechanisms involved remain unclear. Piezo1, a mechanosensitive ion channel expressed on vascular smooth muscle cells (VSMCs), detects mechanical cues and contributes to calcification. Although a link between matrix stiffness, Piezo1 activation, and calcification has been reported, it is uncertain whether increased stiffness promotes osteogenic trans-differentiation and calcification in VSMCs through Piezo1. This study investigates whether elevated vascular matrix stiffness promotes VC by activating Piezo1. Using spontaneously hypertensive rats (SHRs, representing high stiffness) and gelatin methacryloyl (GelMA) hydrogels with variable stiffness (5 %-10 %), we assessed Piezo1's role in stiffness-induced calcification, modulating its activity with the specific agonist Yoda1 and antagonist GsMTx4, and further employing lentivirus-mediated Piezo1 knockdown. Compared to controls, SHRs and high-stiffness GelMA groups exhibited greater calcification (via Alizarin Red staining and RUNX2/BMP2 expression), elevated pulse wave velocity, and increased Piezo1 expression. Pharmacological Piezo1 activation by Yoda1 exacerbated calcification, whereas its inhibition by GsMTx4 markedly attenuated this process-particularly under high-stiffness conditions. Genetic Piezo1 knockdown under calcifying conditions reduced calcium deposition, downregulated BMP2/RUNX2, restored SM22α, and attenuated calcium influx. Notably, the procalcific effect of Yoda1 was abolished in Piezo1-knockdown cells. Additionally, p53/p21, γH2AX, and IL-1β levels rose in parallel with calcification. These findings demonstrate that matrix stiffness promotes VC via Piezo1 activation, identifying Piezo1 as a central mechanotransducer linking vascular stiffness to pathological calcification. Moreover, they provide integrated pharmacological and genetic evidence supporting Piezo1 as a therapeutic target in VC.