Abstract 652: In Vitro Implication Of Mechanisms Mediating Ultrasound-enhanced Vascular Permeability

Background: We have previously shown modeling of ultrasound (US) enhancement of echogenic liposome (ELIP) penetration into the arterial wall using a transwell culture system featuring a human umbilical vein endothelial cell (HUVEC) monolayer. We found the effect was due to US radiation pressure rather than cavitation, mediated by nitric oxide (NO) signaling, and that it did not exclusively require cGMP production. We have now conducted an initial investigation to identify mechanisms mediating this effect. Hypothesis: US radiation pressure impacts the cell-surface receptor involved in blood pressure compensation and shear stress-induced NO production, mediated by Akt phosphorylation. Methods: HUVEC were grown to confluence in a 6-well culture plate, which was subjected to continuous-wave US (Sonitron) for various times from 0 to 50 seconds. Following an additional 30 minutes of incubation at 37 o C, cells in each well were harvested with 100 μl lysis buffer. Lysates were subjected to SDS-PAGE and Western blotting with primary antibodies, followed by secondary antibodies coupled to IR dyes. Blots were visualized with the LI-COR Odyssey Imaging System and quantitated by densitometric analysis with NIH ImageJ software. Results: US treatment of HUVEC caused phosphorylation of Akt and ERK 1/2, at an optimum at 30 seconds of treatment (Fig. 1). Conclusions: The hypothesis was supported, implicating shear stress-induced stimulation of the mitogen-activated protein kinase cascade, involving activation of both Akt and ERK 1/2. In the future, we will investigate pathway initiation and endpoints.