Early Assessment of Atherosclerotic Lesions and Vulnerable Plaques in vivo by Targeting Apoptotic Macrophages with AV Nanobubbles

Abstract

Xiaoju Ma,1,2,* Jia Wang,1,* Zhelong Li,1 Xueying Zhou,3 Xiao Liang,1 Junyan Wang,4 Yunyou Duan,1 Ping Zhao1 1Department of Ultrasonic Medicine, Tang Du Hospital, Air Force Medical University, Xi’an, 710038, People’s Republic of China; 2Ultrasonic Department, Lin Tong Rehabilitation and Convalescent Center, Lintong, 710600, People’s Republic of China; 3Ultrasonic Department, Air Force Hospital of Central Theater, Datong, 037006, People’s Republic of China; 4Department of Nuclear Medicine, Tang Du Hospital, Air Force Medical University, Xi’an, 710038, People’s Republic of China*These authors contributed equally to this workCorrespondence: Ping Zhao; Yunyou Duan, Email [email protected]; [email protected]: The early detection of atherosclerotic lesions is particularly important for risk prediction of acute cardiovascular events. Macrophages apoptosis was significantly associated with the degree of AS lesions and especially contributed to plaque vulnerability. In this research, we mainly sought to explore the feasibility of a home-made AV-nanobubbles (NBAV) for visualization of apoptotic macrophages and assessment of atherosclerosis (AS) lesions by contrast-enhanced ultrasound (CEUS) imaging.Methods: NBAV were prepared by “Optimized Thin-Film Hydration” and “Biotin-Avidin-Biotin” methods. Then, the characterization and echogenicity of NBAV were measured and analyzed in vitro. The targeting ability of NBAV to ox-LDL–induced apoptotic macrophages was observed by laser scanning confocal microscope. The ApoE−/− mice mode fed with high fat diet were observed by high-frequency ultrasound, microanatomy and oil red O staining. CEUS imaging in vivo was performed on AS plaques with NBAV and NBCtrl injection through the tail vein in turn in ApoE−/− mice. After CEUS imaging, the plaques were confirmed and analyzed by histopathological and immunological assessment.Results: The prepared NBAV had a nano-scale size distribution with a low PDI and a negative zeta potential. Moreover, NBAV showed an excellent stability and exhibited a significantly echogenic signal than saline in vitro. In addition, we found that NBAV could target apoptotic macrophages induced by ox-LDL. Compared with NBCtrl, CEUS imaging of NBAV showed strong and sustained echo enhancement in plaque area of aortic arch in vivo. Further research showed that NBAV sensitive plaques presented more significant pathological changes with several vulnerable plaque features and abundant TUNEL-positive area.Conclusion: NBAV displayed a sensitive indicator to evaluate apoptotic macrophages, indicating a promising CEUS molecular probe for AS lesions and vulnerable plaques identification.Graphical Abstract: Keywords: atherosclerosis, vulnerable plaques, macrophage, apoptosis, AV-nanobubbles

Keywords

• atherosclerosis
• vulnerable plaques
• macrophage
• apoptosis
• av-nanobubbles