Reversible Ca²⁺ signaling and enhanced paracellular transport in endothelial monolayer induced by acoustic bubbles and targeted microbeads.

Ultrasound and microbubble mediated blood brain barrier opening is a non-invasive and effective technique for drug delivery to targeted brain region. However, the exact mechanisms are not fully resolved. The influences of Ca²⁺ signaling on sonoporation and endothelial tight junctional regulation affect the efficiency and biosafety of the technique. Therefore, an improved understanding of how ultrasound evokes Ca²⁺ signaling in the brain endothelial monolayer, and its correlation to endothelial permeability change is necessary. Here, we examined the effects of SonoVue microbubbles or integrin-targeted microbeads on ultrasound induced bioeffects in brain microvascular endothelial monolayer using an acoustically-coupled microscopy system, where focused ultrasound exposure and real-time recording of Ca²⁺ signaling and membrane perforation were performed. Microbubbles induced robust Ca²⁺ responses, often accompanied by cell poration, while ultrasound with microbeads elicited reversible Ca²⁺ response without membrane poration. At the conditions evoking reversible Ca²⁺ signaling, intracellular Ca²⁺ release and reactive oxygen species played key roles for microbubbles induced Ca²⁺ signaling while activation of mechanosensitive ion channels was essential for the case of microbeads. Trans-well diffusion analysis revealed significantly higher trans-endothelial transport of 70 kDa FITC-dextran for both integrin-targeted microbeads and microbubbles compared to the control group. Further immunofluorescence staining showed disruption of cell junctions with microbubble stimulation and reversible remodeling of many cell junctions by ultrasound with integrin-targeted microbeads. This investigation provides new insights for ultrasound induced Ca²⁺ signaling and its influence on endothelial permeability, which may help develop new strategies for safe and efficient drug/gene delivery in the vascular system.