Abstract 15915: Ultrasound Targeted Microbubble Cavitation-Induced Endothelial Hyperpermeability is Calcium-Dependent and Regulated by Enos Signaling

Abstract

Introduction: Ultrasound ( US )-targeted microbubble ( MB ) cavitation ( UTMC ) facilitates delivery of cell-impermeant drugs across the endothelial barrier; mechanisms are poorly understood. We reported that UTMC causes inter-endothelial gaps in cultured endothelial cell ( EC ) monolayers, which could increase barrier permeability, and that UTMC increases Ca 2+ influx into ECs. Given that increased intracellular Ca 2+ activates eNOS, and nitric oxide (NO) release promotes microvascular permeability, we hypothesized that UTMC increases intracellular Ca 2+ through activation of mechanosensitive Piezo-1 Ca 2+ channels, leading to NO synthesis and endothelial barrier hyperpermeability. Methods: Transwells were endothelialized with human coronary artery ECs. Lipid MBs were added, and US was delivered for 10 s (1 MHz, 250 kPa, 10 cycles, 10 ms interval). Permeability was measured using dextran transfer and transendothelial electrical resistance ( TEER ). GsMTx4 and L-NAME were used to inhibit Piezo1 and eNOS, respectively; Fluo-4 AM to visualize Ca 2+ influx; DAF-FM to detect NO. S-nitrosylation was measured with biotin switch assay. Results: UTMC reduced TEER (1.7 fold, p<0.05 ) and increased dextran flux across the transwell (2-fold, p<0.01 for 70 kDa dextran, 1.6 fold, p<0.05 for 10 kDa dextran), which was abrogated by L-NAME. UTMC significantly increased Ca 2+ influx into ECs and tripled NO ( p<0.05 ), but these increases and UTMC-mediated hyperpermeability were blunted by Piezo1 channel inhibition ( p<0.0001) . UTMC increased S-nitrosylation of proteins (1.7 fold, p<0.01 ) and was associated with s-nitrosylation of VE-cadherin, its redistribution into focal adhesion junctions, and increased stress fiber formation, which were abolished by L-NAME. Conclusions: UTMC causes endothelial barrier hyperpermeability through a Ca 2+ -dependent mechanism, where Ca 2+ influx into ECs, mediated in part by mechanosensitive Piezo1 channels, modulates eNOS signaling. Our data suggest that eNOS regulates UTMC-induced hyperpermeability, possibly through s-nitrosylation of VE-cadherin. Further studies to examine mechanisms regulating UTMC bioeffects will aid in clinical translation and optimization of UTMC for delivery of cell-impermeant drugs.