טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentChen Geffen
SubjectThe Effect of Low Intensity Ultrasound on Adhesion
Molecules, Actin Monomers and Membrane
Permeability in Endothelial Cells
DepartmentDepartment of Biomedical Engineering
Supervisor Full Professor Kimmel Eitan
Full Thesis textFull thesis text - English Version


Abstract

Biological effects induced by low intensity ultrasound (US) in cultured endothelial cells (ECs) in vitro have been traditionally attributed to microstreaming that is associated with bubble activity (cavitation) and to radiation pressure induced streaming, and to the shear stresses that act on the cells. Lately, a novel concept of intramembrane cavitation was suggested as a leading mechanism for the influence of US on ECs. In our experimental setup, an US beam was aimed from underneath a horizontal cell culture dish, which is coated with ECs monolayer at the bottom. Acoustic pressure amplitudes in the range between 50 and 300 kPa were tested using 1MHz frequency (20% duty cycle) for 5 min. A tracer spreading in the plate under US showed that in our system, in the medium surrounding the cells, the streaming is rather weak and microstreaming is negligible. Then, we examined three elements in the ECs that are known to be affected by mechanical forces: i) membrane permeability, ii) αVβ3 integrin activation and iii) β-actin monomers. We demonstrated that US significantly increased membrane permeability of ECs. The integrin clustering in response to US was hard to determine because in acoustic pressure amplitude of 200 kPa and above the cells were torn off the dish surface. There was no significant difference in the expression of β-actin monomers at various US pressure amplitudes, even microbubbles (ultrasound contrast agents) were added to the medium above the ECs. In absence of streaming related shear stress on the ECs’ surface, it seems that the results can be explained by the effects of the oscillating acoustic pressure on the bilayer membrane expansion and increase in tension as described in the new model. We speculate that when the cultured ECs were exposed to the US two opposing mechanisms occurred simultaneously: the increase in the membrane permeability and the cell rupture on one hand which caused actin monomer leakage and integrin detachment from ruptured membranes; and the mechanical loading on the other hand which resulted in integrin activation and cytoskeleton reorganization that led to expression of more actin monomers. To conclude, it is reasonable to assume based on the intramembrane cavitation model that the stretching level of the bilayer membrane as well as membrane permeability and cytoskeleton reorganization increase with the US pressure amplitude, and that as the pressure amplitude increase further membrane rupture and detachment of the ECs from the surface are more likely to occur.