ASSA14-15-01 Angiogenesis effect of therapeutic ultrasound on ischaemic hind limb in mice

Abstract

Background Therapeutic ultrasound (TUS) has been proposed as a potential strategy for therapeutic revascularisation. However, the specific mechanism remains to be explored in detail. The purpose of this study was to determine the mechanism of TUS on ischaemic angiogenesis in mice with acute ischaemic hind limb and proliferation, migration, tube formation and potential signalling pathways of human umbilical vein endothelial cells (HUVECs). Methods TUS was set by a combination of various ultrasound-related variables: frequency of 1.0 MHz; intensity of 0.3 W/cm2; exposure time of 3, 6, or 9 min. We stimulated the HUVECs in culture daily with TUS for 3 days. The effects of therapeutic ultrasound on HUVECs proliferation were evaluated using the CCK-8 assay. Cell migration was assayed by scratch test. Angiogenesis was observed by tube formation assay. The potential involvement of PI3K-Akt-eNOS signalling pathways was explored using selective chemical inhibitor or Western-blot analysis. Left femoral artery ligation was used to establish the model of acute hind limb ischaemic in mice (C57BL/6J male mice, 18–22 g, 2–3 months old), TUS was exposed subcutaneously once everyday for 14 days. The mice were randomly divided into four experimental groups: sham operated group; TUS 3 min group; TUS 6 min group and TUS 9 min group. The experiment was carried out on the 1st and14th after ligation. To determine whether TUS affected the angiogenesis activity of mice with ischaemic hind limb, we measured temperature of hind limb by using infrared thermometer, and microvessel density (MVD) was determined by CD31 staining. Results The results show that proliferation, migration and tube formation in HUVECs could be promoted by TUS at exposure time of 9 min, and inhibited by NG-Nitro-L-arginine Methyl Ester (L-NAME). eNOS and p-eNOS expression increased significantly when TUS 9 min was added, but could be attenuated by L-NAME. p-Akt expression augmented by TUS at exposure time of 9 min, but could be inhibited by LY294002. Compared to the temperature of hind limb, the group of TUS 9 min at the 14th was increased, while TUS 6 min group increased, but only TUS 9 min was there a significant difference. Except that, the MVD was promoted in TUS 9 min group significantly. Conclusions In conclusion, the present study indicates that 9 min of exposure to TUS promote angiogenesis among ischaemic hind limb in mice and that this process is mediated through the PI3K-Akt-eNOS signal pathway in ECs.