Effect of high power ultrasound on endothelial cells--an in vitro study of the endothelium--hemostasis interaction.

The goal of this study was to develop an in vitro model system in which the hemostatic effects of high power ultrasound applied to the outer surface of blood vessels during tumor dissection can be simulated and measured.Monolayers of endothelial cells (HUVEC, ATCC) in cell culture plates were sonicated with an ultrasound dissector (SONOCA II, Soering) at a frequency of 23.5 kHz. The dissector was equipped with a cooling circuit. The cell cultures were exposed to 2 minutes of continuous ultrasound with intensities of 10, 50, or 100 W/cm(2). To differentiate between heat and sound effects, selected monolayers were warmed for 2 minutes. Finally, the cell cultures were stained with trypan blue to assess for cell death due to membrane disruption. Cytomorphological alterations and changes in the concentration of coagulation parameters in the cell culture medium were evaluated.The cytomorphological alterations were found to depend on ultrasound intensity. They included detachment of single endothelial cells, cell cluster formation and cytoplasmic cavitation. Disruption of the cell membrane integrity was infrequently observed. Of 14 screened coagulation parameters, thromboxane B(2) (TXB(2)), prostaglandin F(1alpha) (PGF(1alpha)), plasminogen activator inhibitor type 1 (PAI-1), thrombomodulin (TM), and thrombospondin (TSP) were found to be ultrasound sensitive. TXB(2) concentrations in the medium increased beginning at low ultrasound intensities (p < 0.01) and were independent of temperature. PGF(1alpha) concentrations peaked at high ultrasound intensities (p < 0.05), and heat alone produced a significant increase in concentration (p < 0.05). At high intensities, the ratio of TXB(2) to PGF(1alpha) shifted in favour of PGF(1alpha). PAI-1 was most strongly secreted at low ultrasound intensities (p < 0.01), and heat resulted in a decrease of concentration (p < 0.05). TM and TSP concentrations correlated strongly and reached a non significant peak at low intensities.The results demonstrate that during sonication of endothelial cells in vitro, coagulation parameters are released from distant undamaged cells. HUVEC-cells exhibit a differential hemostaseological response at different ultrasound intensities, and the response is also influenced by heat. Additionally, massive morphological damage can be induced at the endothelium.