Diagnostic ultrasounds activate caveolar internalization and induce phosphorylation of Erk ½ and eNOS in human endothelial cells

Abstract

Diagnostic ultrasound (US) can markedly enhance the endothelial protein uptake in vivo. However, the mechanisms are still unknown. Since caveolae play a major role in cellular endocytosis, we hypothesized that US activate caveolar trafficking. As a marker of caveolar trafficking, we used the recombinant glutathione S-transferase (GST)-Tat11-EGFP fusion protein. Cultured human umbilical vein endothelial cells (HUVEC) were incubated for 90′, before being exposed to US, with 2μg/ml of (GST)-Tat11-EGFP or GST-EGFP, a marker of endothelial plasma membrane integrity. HUVEC were analyzed by flow cytometry after sham (transducer off) exposure to US and after 30′ of US irradiation with second harmonic 1.3/2.6 MHz cardiac US scan (mechanical index 1.2). Furthermore, we measured by Western blot the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERKErk ½) and endothelial nitric oxide synthase (eNOS),which is known to be coupled with caveolar trafficking pathway. Flow cytometry showed a significant enhancement of GST-Tat11-GFP uptake after 30′ of US exposure in treated cells compared to sham (9.0±1.0 vs 4.5±0.5 A.U., P≤0.05), but not GST-GFP. Moreover, US induced phosphorylation of ERK½(12-fold,P≤0.05) and eNOS ser-1177 (15-fold, P≤0.05) compared to sham. In conclusion,cardiac diagnostic US increase the endothelial protein uptake by activation of caveolar trafficking without disruption of cell membrane integrity, associated with the phosphorylation/activation of ER½and eNOS.