Shock wave effects on the vascular endothelium

Detta är en avhandling från Stockholm : Karolinska Institutet, Karolinska Institutet, Stockholm Söder Hospital

Sammanfattning: A shock wave is a pressure wave with high amplitude and a velocity above sound. Man is exposed to shock waves (SWs) in connection with trauma, e.g. explosions and high-energy missiles and medical treatments such as extracorporeal lithotripsy (ESWL). A major consequence of SWs is blood vessel injury. The ensuing detachment of endothelial cells (EC) and rupture of basal membranes partially explain the hemorrhages, thrombus formation and tissue edema, seen after SW exposure. Additional effects on endothelial cell functions are, however, conceivable in order to explain the panorama of injury, which might proceed to multiple organ failure. The flyer-plate technique is an established method to detonate explosives. This thesis describes how the flyer-plate technique was modified to expose cell monolayers to precise SWs, in ordinary cell culture wells. Mean peak amplitudes generated were 23 MPa or 104 MPa, depending on the experimental setting. Human umbilical vein endothelial cells (HUVECs) were used for cell experiments. To elucidate the mechanisms involved in SW injury we assessed the role of two suggested factors, viz. cavitation bubbles and reactive oxygen species (ROS). HUVECs exposed to SWs only, could not be distinguished from controls with regards to morphology or cell viability. Yet, a sub-lytic cell membrane injury was indicated. HUVEC cultures exposed to SWs plus cavitation (SWC) exhibited discrete endothelial lesions. Within the lesion area cell detachment, cell membrane damage and cell death was observed. A dose- response relationship between cavitation and cell injury was demonstrated. No protective effects of ROS scavengers were shown. To gain further insight into the pathophysiology of SW tissue injury we assessed the effects of SWs or SWC on the adhesion molecules P-selectin, E-selectin and ICAM-1 as well as cytoskeletal organization. A previous study had shown that H2O2 can induce reversible disassembly of tubulin and vimentin filaments. No significant effects of SWs per se, on the studied parameters, were observed. A sequential induction of P- selectin, E-selectin and ICAM-1 expression was shown in samples exposed to SWC. The induction of E- selectin and ICAM-1 was preceded by nuclear translocation of the NFkappaB subunit p65, implicating transcriptional regulation of this response. Destruction of actin, tubulin and vimentin filaments was seen in cells within the main lesion area. Cells peripheral to this area displayed disassembly of dense peripheral bands and formation of actin stress fibers, implying enhanced monolayer permeability in this region. Finally, we studied the cytoskeletal reorganization in relation to endothelial regeneration after SWC injury. It was shown that endothelial lesions, caused by SWC, exhibited impaired endothelial regeneration rate compared to mechanically produced lesions used as controls. This SW effect could be related to hampered cytoskeletal redistribution of tubulin- and actin filaments at the lesion border. In conclusion the flyer-plate technique can be used to expose cell monolayers to reproducible SWs. Our experimental model separates the effects of SWs from the effects of SWC. SWs per se caused cell membrane injury without affecting cell viability. Endothelial cell detachment and lytic cell injury, previously described in relation to explosions, high-energy missile trauma, laser angioplasty, ophthalmologic laser surgery and ESWL, was, however, linked to the generation of cavitation. Finally, SWC lesions were associated with a pro-inflammatory reaction as well as impaired endothelial regeneration rate, which may be attributed to changes in cytoskeletal functions.

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