To investigate the effect of annexin V conjugated nanobubbles (NB_AV) on atherosclerotic (AS) lesions, tissue factor (TF) expression in atherosclerotic plaques, and lipid levels in ApoE ^-/- mice, and explore the potential value of NB_AV in plaque intervention, as well as their anti-thrombotic effects.

ApoE ^-/- mice (n=50) were fed a high fat for 8~10 weeks to establish an atherosclerotic plaque model, and normal C57 mice (n=10) were uesed as controls. The characterization of plaques was performed by high-frequency ultrasound, microanatomy, and oil red O staining. In vivo contrast-enhanced ultrasound (CEUS) was then performed on AS plaques in ApoE ^-/- mice with NB_AV and NB_Ctrl injection. After CEUS, the plaques were histopathologically assessed in vitro by hematoxylin-eosin, Masson, oil red O, and TUNEL staining. Model mice were randomly divided into a nontreatment group (n=20) and a treatment group (n=20). The treatment group was injected with 150 μl of NB_AV dilution (50 μl/10 g) via the tail vein every 5 days for 8 weeks. The nontreatment group was given the same amount of normal saline. After several weeks of treatment, blood vessel samples were collected for analysis of TF expression and blood samples were collected for blood lipid analysis.

After 8 weeks of high fat feeding, ApoE ^-/- mice showed echo-enhanced atherosclerotic plaques, as revealed by microscopic findings and oil red O staining. Compared with mice injected with NB_Ctrl, in vivo CEUS showed that there was strong and sustained echo enhancement in plaque area of the aortic arch in mice with NB_AV injection [60 s: (106.33±23.0) dB vs (62.33±18.5) dB; 120 s: (101.33±17.3) dB vs (41.67±10.3) dB; 180 s: (72.67±10.2) dB vs (22.00±5.2) dB, P < 0.01]. Further histopathological results showed that plaques in mice with NB_AV injection presented significant pathological changes with vulnerable features and abundant TUNEL-positive area, such as irregular morphology, disordered arrangement of the vessel wall, a large amount of lipid accumulation, and a decrease in blue-dyed collagen fibers in the plaque. The positive TF staining areas in plaques began to decrease after 6 weeks of NB_AV treatment, and TF expression was significantly different compared with that at 0 and 2 weeks of NB_AV treatment (t =14.17, P < 0.001; t =7.022, P < 0.01). The levels of TG, TC, and LDL-c were significantly lower in mice treated with NB_AV for 8 weeks than in nontreatment group (P < 0.05), while the level of HDL-c was significantly higher than that of the nontreatment group (P < 0.05).

NB_AV can be used to evaluate AS plaque vulnerability and reduce TF expression within plaques and blood lipids, suggesting that NB_AV have potential value in intervening in AS lesions.