The effects of ultrasound on endothelial cell behaviour and angiogenesis

Shriane, Lisa (2026). The effects of ultrasound on endothelial cell behaviour and angiogenesis. University of Birmingham. Ph.D.

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Abstract

Ultrasonic surgical devices working at kHz frequencies are used to cut hard and soft tissues. Such precision instruments facilitate access and visualisation during surgery and the ultrasound may also promote healing. The biological effects of ultrasound exposure have been reported in vitro and in vivo and represents a developing field of research. However, the effects of low frequency kHz ultrasound on cells remains under-researched. The aim of the present work was to study the effects of low frequency ultrasound on angiogenesis which is essential for initial wound healing and subsequently post-surgical healing.

Human umbilical vein endothelial cells (HUVECs) were exposed to 45 kHz ultrasound for 5 min using a DuoSon commercial device. The angiogenic response was analysed via F-actin staining, cell counting, a tube formation assay and qPCR techniques at multiple time points. The HUVEC cytoskeleton was observed to be largely unaffected after DuoSon exposure. Significant increases in viable cell number were measured 72 h after 25 mW/cm2 (27 %, p < 0.001) and 75 mW/cm2 (25 %, p < 0.001) exposure compared with untreated cells. This stimulatory effect was not observed in the tube formation ability of HUVECs, however a time-dependent effect was apparent. Ultrasound-treated HUVECs retained tube formation ability for longer during the in vitro assay compared with untreated cells when analysed 72 h after exposure. Exposure to 75 mW/cm2 also resulted in significantly reduced eNOS expression (p < 0.05) compared with untreated cells 6 h after exposure. The expression levels were comparable with untreated cells by 72 h. This indicated an early effect on gene expression that returned to control levels by 72 h in contrast to the viable cell numbers.

The DuoSon exposure system provided initial observations of a cellular response to 45 kHz ultrasound. However the exposure set up did not allow measurement of the ultrasound delivered to cell monolayers. Furthermore, the levels of ultrasound would be increased by reflections at multiple interface boundaries in the set up used. To overcome such difficulties, a standardised in vitro acoustic absorbing tank exposure system was designed. An acoustically transparent CLINIcell® cassette was placed in a degassed water-filled tank lined with ultrasound absorbing tiles. This demonstrated a reduction of ultrasound reflections within the acoustic field during pressure field scanning. The exposure system also allowed thermal control of the water. HUVECs exposed to 45 kHz appeared with non-uniform morphology in certain instances 72 h after exposure. Cell viability was unaffected, though no significantly damaging effect in viable cell number was observed as seen with the DuoSon analysis. Interestingly, qPCR revealed a significant downregulation (p < 0.05) in eNOS 72 h after exposure compared with downregulation observed 6 h after DuoSon delivery.

In conclusion, this work demonstrated an effect of 45 kHz ultrasound on HUVEC behaviour. HUVEC viability was not significantly damaged, rather an increase in viable cell number was measured. An apparent ability to retain tube formation ability was also observed. Significant intracellular effects were observed, specifically a downregulation in eNOS expression. A visible effect of ultrasound was observed in some instances of altered cell shape which may provide reasoning for the cellular responses measured. The acoustic absorbing tank developed in this study provides researchers with a standardised exposure method for delivery of ultrasound in vitro. This will allow improved understanding of the biological effects of ultrasound which in turn will assist in the future development of ultrasonic surgical instruments and other medical devices.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):