In Vivo Validation of Modulated Acoustic Radiation Force–Based Imaging in Murine Model of Abdominal Aortic Aneurysm Using VEGFR-2–Targeted Microbubbles
Authors: Huang Yi , Herbst Elizabeth B. , Xie Yanjun , Yin Li , Islam Zain H. , Kent Eric W. , Wang Bowen , Klibanov Alexander L. , Hossack John A. 
Published: July 2023

Objectives
The objective of this study is to validate the modulated acoustic radiation force (mARF)–based imaging method in the detection of abdominal aortic aneurysm (AAA) in murine models using vascular endothelial growth factor receptor 2 (VEGFR-2)–targeted microbubbles (MBs).

Materials and Methods
The mouse AAA model was prepared using the subcutaneous angiotensin II (Ang II) infusion combined with the β-aminopropionitrile monofumarate solution dissolved in drinking water. The ultrasound imaging session was performed at 7 days, 14 days, 21 days, and 28 days after the osmotic pump implantation. For each imaging session, 10 C57BL/6 mice were implanted with Ang II–filled osmotic pumps, and 5 C57BL/6 mice received saline infusion only as the control group. Biotinylated lipid MBs conjugated to either anti–mouse VEGFR-2 antibody (targeted MBs) or isotype control antibody (control MBs) were prepared before each imaging session and were injected into mice via tail vein catheter. Two separate transducers were colocalized to image the AAA and apply ARF to translate MBs simultaneously. After each imaging session, tissue was harvested and the aortas were used for VEGFR-2 immunostaining analysis. From the collected ultrasound image data, the signal magnitude response of the adherent targeted MBs was analyzed, and a parameter, residual-to-saturation ratio (R

res − sat
), was defined to measure the enhancement in the adherent targeted MBs signal after the cessation of ARF compared with the initial signal intensity. Statistical analysis was performed with the Welch t test and analysis of variance test.

Results
The R

res − sat
of abdominal aortic segments from Ang II–challenged mice was significantly higher compared with that in the saline-infused control group (P < 0.001) at all 4 time points after osmotic pump implantation (1 week to 4 weeks). In control mice, the R

res − sat
values were 2.13%, 1.85%, 3.26%, and 4.85% at 1, 2, 3, and 4 weeks postimplantation, respectively. In stark contrast, the R

res − sat
values for the mice with Ang II–induced AAA lesions were 9.20%, 20.6%, 22.7%, and 31.8%, respectively. It is worth noting that there was a significant difference between the R

res − sat
for Ang II–infused mice at all 4 time points (P < 0.005), a finding not present in the saline-infused mice. Immunostaining results revealed the VEGFR-2 expression was increased in the abdominal aortic segments of Ang II–infused mice compared with the control group.

Conclusions
The mARF-based imaging technique was validated in vivo using a murine model of AAA and VEGFR-2–targeted MBs. Results in this study indicated that the mARF-based imaging technique has the ability to detect and assess AAA growth at early stages based on the signal intensity of adherent targeted MBs, which is correlated with the expression level of the desired molecular biomarker. The results may suggest, in very long term, a pathway toward eventual clinical implementation for an ultrasound molecular imaging–based approach to AAA risk assessment in asymptomatic patients.