The activation of endothelial nitric oxide synthase, induced by calcium influx plays a crucial role in regulating endothelial hyperpermeability caused by ultrasound-targeted microbubble cavitation

Abstract

Ultrasound-targeted microbubble cavitation (UTMC) plays a crucial role in improving drug delivery across the endothelial barrier. For successful application of UTMC in clinic, an understanding of molecular mechanisms involved is essential. Here, we hypothesized that Ca2+ and endothelial nitric oxide synthase (eNOS) pathways regulate UTMC-induced endothelial hyperpermeability. We used human coronary artery endothelial cells seeded on transwell inserts, exposed to microbubbles and ultrasound (frequency 1 MHz; PNP 250 kPa; pulse length 10 μs; pulse interval 10 ms; treatment duration 10 s). UTMC caused cellular influx of Ca2+, which was inhibited by blocking mechanosensitive channels (p < 0.0001) using GsMTx4. Knockdown of Piezo1 using siRNA showed similar effects. UTMC-induced Ca2+ influx activated eNOS and enhanced nitric oxide production, which was necessary for UTMC-induced hyperpermeability. Our data suggest that UTMC induces a Ca2+ influx-dependent increase in S-nitrosylation: An increase in S-nitrosylation was observed on both β-catenin and VE-cadherin after UTMC, potentially contributing to the destabilization of adherens junctions that normally maintain barrier integrity. Our study explains the role of Ca2+ influx, eNOS activation, and enhanced S-nitrosylation of adherens junction proteins in the regulation of endothelial hyperpermeability. Further investigation of these pathways will aid in clinical translation and optimization of UTMC for delivering cell-impermeant drugs.