Enhanced caveolae-mediated endocytosis by diagnostic ultrasound in vitro

The modulation of cellular endothelial permeability is a desirable goal for targeted delivery of labels and therapeutic macromolecules; the underlying mechanisms, however, remains poorly understood. Here, we hypothesize that a higher endothelial permeability may result as an outcome of selective enhancement of caveolar endocytosis by ultrasound (US), in the frequency and intensity range of current clinical diagnostic use. To assess the role of free radicals in this phenomenon, we exposed confluent human endothelial cells to pulsed diagnostic US for 30 min, with a mechanical index (MI) of 0.5 and 1.2, using a 1.6-MHz cardiac US scan, and endothelial cells not exposed to US were used as control. Here we show that pulsed diagnostic US with a MI of 1.2 (high mechanical index ultrasound [HMIUS]) were able to selectively enhance endothelial caveolar internalization of recombinant glutathione-S-transferase (GST)-Tat11-EGFP fusion protein (26 +/- 1 vs. 11.6 +/- 1 A.U, p < 0.001 vs. control), without disruption of plasma membrane integrity. Moreover, pulsed diagnostic US with a MI of 0.5 (low mechanical index ultrasound) did not increase caveolar endocytosis compared with control (11.4 +/- 1.2 vs. 11.6 +/- 1). Free-radical generation inhibitors, such as catalase and superoxide dismutase, reduced the HMIUS-induced caveolar internalization by a 49.29% factor; finally, HMIUS-induced caveolar endocytosis was found to be associated with a significant increase in the phosphorylation of tyr-14-caveolin1, ser1177-eNOS and Thr202/Tyr204-ERK(1/2) compared with control. These findings show how HMIUS irradiation of human endothelial cells cause a selective enhancement of caveolar-dependent permeability, partially mediated by free radicals generation, inducing a marked increase of phosphorylation of caveolar-related proteins. Thus, the use of diagnostic US could potentially be used as an adjuvant to drive caveolar traffic of extracellular peptides by using a higher level of US energy.