Ultrasound-targeted microbubble cavitation-induced Ca2+ influx via Piezo1 causes transient gene expression changes

Background: Ultrasound-targeted microbubble cavitation (UTMC) is a viable approach to overcome challenges in efficient targeted drug delivery by inducing transient endothelial hyperpermeability. However, a deeper understanding of molecular mechanisms and bioeffects of UTMC is essential for clinical translation. Methods and results: Using human coronary artery endothelial monolayers on transwells, we found that UTMC (f 1 MHz; PNP 250 kPa; pulse length 10 μs; pulse interval 10 ms; treatment duration 10 s) caused Ca2+ influx, which was required for inter-endothelial gap formation and hyperpermeability. Inhibitor (GsMTx4) and siRNA studies revealed that a significant fraction of Ca2+ influx occurred via the mechanosensitive channel Piezo1. To determine gene expression changes following Ca2+ influx via Piezo1, we performed RNA-seq in GsMTx4-treated cells post-UTMC. Pathway analysis predicted activation of multiple hyperpermeability- and inflammation-related pathways, including HIF1α, STAT3, ERK/MAPK, Notch, CXCR4, NO, RHO GTPase, and IL-4, 7, 8, 9, 13 signaling, at 3 h post-UTMC, returning to baseline by 24 h. A preliminary study using specific inhibitor WZ811 suggested that CXCR4 might play a key role in regulating UTMC-induced hyperpermeability. Conclusions: Our findings provide important new insights into the mechanisms underlying UTMC-induced hyperpermeability and potential bioeffects, paving the way for optimizing this technology for safe and effective clinical application.