M.Sc Thesis

M.Sc StudentMizrahi Natalya
SubjectThe Influence of Therapeutic Ultrasound on Angiogenic
Receptors and Endothelial Cells Proliferation and
Migration in Vitro
DepartmentDepartment of Biomedical Engineering
Supervisor PROFESSOR EMERITUS Eitan Kimmel


Angiogenesis is the complex process by which new blood vessels arise from the pre-existing vasculature. Therapeutic angiogenesis is the controlled induction or stimulation of new blood vessel formation to reduce unfavorable tissue effects caused by local hypoxia and to enhance tissue repair. Ultrasound has been used for over 40 years to stimulate repair. The effects of ultrasound on wound healing, chronic ulcers and fracture healing may be explained by the enhancement of angiogenesis.

            The objective of this project is to evaluate the influence of low intensity TUS (intensity up to 2W/cm2 and frequencies 1 and 1.5 MHz) on cultured EC with respect to angiogenesis phenomena.

The estimation of Flk-1 receptor expression and regulation due to TUS irradiation was performed using immunobloting and immunofluorescence techniques. Evaluation of EC proliferation rate was done by recording cells proliferation in a period of 12 hours after TUS treatment. EC migration and sprouting were estimated using a 3D cell structure of endothelial spheroids that was embedded in collagen and PEG-fibrinogen gel matrix. The morphology of the spheroid-like structures was observed using optic microscope and histological slices. The results suggest that exposure of EC to low intensity TUS affects EC phenotype by downregulation of Flk-1 expression. Reduction in Flk-1 receptor concentration is observed immediately after TUS treatment and additional decrease is measured after 12 hours incubation. TUS treatment also stimulates translocation of Flk-1 receptor on the EC membrane from the periphery to a zone close to the nucleus. In addition, TUS stimulates EC proliferation by reducing cell cycle time constant that was estimated using cell proliferation model. Moreover the rate of sprouting and cell migration, observed in endothelial spheroids, intensifies due to TUS treatment. A stable, sprout like structures appear in TUS treated EC spheroids on the second day of incubation, while similar sprouts appear in untreated spheroids only after the third day. However irradiated spheroids become unstable and capillary-like structures break after three days of incubation. In difference untreated spheroids develop a stable capillary like sprouts at the seventh day of incubation.

This study may help in clarifying how low intensity TUS affects angiogenesis processes and may evolve into a method of targeted stimulation of new blood vessel formation for therapeutic purposes as well as in tissue engineering applications.