Ultrasound-targeted microbubble destruction rapidly improves left ventricular function in rats with ischemic cardiac dysfunction.

BACKGROUND: Previous studies have demonstrated the efficacy of ultrasound-targeted microbubble destruction (UTMD) in the treatment of ischemic heart failure (HF). The purpose of this study was to explore the mechanism by which UTMD improves ischemic HF. METHODS: An ischemic heart failure model was established using Sprague-Dawley rats. Rats were randomly divided into 7 groups: sham group, HF group, HF + MB group, HF + ultrasound (US) group, HF + UTMD group, HF + UTMD+LY294002 group, and HF + LY294002 group. Serum BNP level and echocardiographic parameters were measured to evaluate cardiac function. PI3K/Akt/eNOS signaling pathway protein levels were detected by immunohistochemistry (IHC) and western blotting. The concentrations of nitrous oxide (NO) and ATP were detected by ELISA, and hematoxylin and eosin (HE) staining was used to evaluate myocardial tissue. RESULTS: UTMD rapidly improved ejection fraction (EF) (HF: 37.16 ± 1.21% vs. HF + UTMD: 46.31 ± 3.00%, P < 0.01) and fractional shortening (FS) (HF: 18.53 ± 0.58% vs. HF + UTMD: 24.05 ± 1.84%, P < 0.01) in rats with ischemic HF. UTMD activated the PI3K/AKT/eNOS signaling pathway (HF vs. HF + UTMD, P < 0.01) and promoted the release of NO and ATP (HF vs. HF + UTMD, both, P < 0.05). Inhibition of the PI3K/AKT/eNOS signaling pathway by LY294002 worsened EF (HF: 37.16 ± 1.21% vs. HF + LY294002: 32.73 ± 3.05%, P < 0.05), and the release of NO and ATP by UTMD (HF + UTMD vs. HF + UTMD+LY294002, P < 0.05). CONCLUSIONS: UTMD can rapidly improve cardiac function in ischemic HF by activating the PI3K/Akt/eNOS signaling pathway and promoting the release of NO and ATP.

Sono-assisted-thrombolysis by three-dimensional diagnostic ultrasound improves epicardial recanalization and microvascular perfusion in acute myocardial infarction.

Background: This study aimed to evaluate the multiple interactions between therapeutic ultrasound (TUS), microbubbles (MB), and recombinant tissue plasminogen activator (r-tPA) by using three-dimensional (3D) ultrasound to examine the impact of thrombolysis with r-tPA on epicardial recanalization and microcirculation in patients with acute ST-segment-elevation myocardial infarction (STEMI). Methods: Acute thrombotic occlusion of the left anterior descending (LAD) artery was induced in 32 Bama pigs, who were fed a high-cholesterol diet and randomized into four groups: (I) a 3D-sono-assisted-thrombolysis (3D/TUS + MB + r-tPA) group; (II) a 3D/TUS + MB group; (III) a full-dose r-tPA group; and (IV) a 3D/TUS alone group. Epicardial angiographic recanalization rate, microcirculation in the at-risk myocardium, ST-segment elevation on electrocardiogram, and changes in the at-risk myocardium and the myocardial infarct area were compared between the groups. Results: After treatment, distal LAD recanalization was observed in 87.5% (7/8) of pigs in the 3D/TUS + MB + r-tPA group, which was significantly higher than the rates observed in the 3D/TUS + MB (37.5%) and the full-dose r-tPA (50.0%) groups (all P<0.05). The average acoustic intensity in the 3D/TUS + MB + r-tPA group (193.78±10.15 dB) was also significantly higher than that in the 3D/TUS + MB (154.29±31.94 dB) and the r-tPA (141.42±28.31 dB) groups (all P<0.05). The decrease in ST-segment elevation in the 3D/TUS + MB + r-tPA group (1.31±1.22 mm) was significantly higher than that in the 3D/TUS + MB (5.38±1.77 mm) and the r-tPA (4.30±2.08 mm) groups (all P<0.05). Furthermore, the ratio of the infarcted myocardial area divided by the at-risk myocardial area was markedly lower in the 3D/TUS + MB + r-tPA group (0.51±0.14) than in the 3D/TUS + MB (0.69±0.28) and r-tPA (0.75±0.23) groups (all P<0.05). Conclusions: Three-dimensional sono-assisted-thrombolysis directly improves infarct-related recanalization rates, enhances microcirculation, reduces r-tPA dosage, and ameliorates the thrombolytic effect of r-tPA in acute STEMI.

Ultrasound-Stimulated Microbubbles Enhance Radiosensitization of Nasopharyngeal Carcinoma.

BACKGROUND/AIMS: Recent studies indicate that therapies targeting the vasculature can significantly sensitize tumors to radiation. Ultrasound-stimulated microbubbles (USMBs) are regarded as a promising radiosensitizer. In this study, we investigated the effect of USMBs on the sensitivity of nasopharyngeal carcinoma (NPC) to radiation. METHODS: Human NPC (CNE-2) cells and human umbilical vein endothelial cells (HUVECs) were exposed to radiation (0, 2, and 8 Gy) alone or in combination with USMBs. Cell viability and apoptosis were measured with the MTT assay and flow cytometry, respectively. The angiogenic activity of HUVECs was detected using matrigel tubule formation. The in vitro effects induced by these treatments were confirmed in vivo with xenograft models of CNE-2 cells in nude mice by examining vascular integrity using color Doppler flow imaging and cell survival using immunohistochemistry. Additionally, the in vivo and in vitro expressions of angiotensin II (ANG II) and its receptor (AT1R) were detected by immunohistochemistry and western blotting, respectively. With CNE-2 cells and HUVECs transfected with control, ANG II, or AT1R, perindopril (an inhibitor of angiotensin-converting enzyme) and candesartan (an inhibitor of AT1R) were used to verify the role of ANG II and AT1R in the radiosensitivity of tumor and endothelial cells by USMBs, by determining cell viability and apoptosis and angiogenic activity. RESULTS: In the NPC xenografts, USMBs slightly reduced blood flow and CD34 expression, increased tumor cell death and ANG II and AT1R expression, and significantly enhanced the effects of radiation. With CNE-2 cells and HUVECs, the USMBs further enhanced the inhibition of tumor cell viability and endothelial tubule formation and further enhanced the increase in ANG II and AT1R due to radiation. Furthermore, perindopril and candesartan significantly enhanced the inhibitory effect of radiation and USMBs on tumor cell growth and angiogenesis in vitro. CONCLUSIONS: We have demonstrated for the first time that USMB exposure can significantly enhance the destructive effect on NPC of radiation, and this effect might be further increased by ANG II and AT1R inhibition. Our findings suggest that USMBs can be used as a promising sensitizer of radiotherapy to treat NPC, and the clinical effect might be increased by ANG II and AT1R inhibition.

Ultrasound molecular imaging of arterial thrombi with novel microbubbles modified by cyclic RGD in vitro and in vivo.

Despite immense potential, ultrasound molecular imaging (UMI) of arterial thrombi remains very challenging because the high-shear arterial flow limits binding of site-targeted microbubbles to the thrombi. The linear Arg-Gly-Asp (RGD) peptides have been successfully applied to evaluate venous, atrial, and arteriolar thrombi, but have thus far failed in the detection of arterial thrombi. Cyclic RGD (Arg-Gly-Asp-D-Phe-Cys) is a cyclic conformation of linear RGD peptides, which has much higher binding-affinity and selectivity for binding to the glycoprotein (GP) IIb/IIIa receptor than its linear counterpart and thus is likely to be an optimal targeted molecular probe for ultrasound molecular imaging of arterial thrombi. In this study, we sought to assess the feasibility of a novel microbubble conjugated with cyclic RGD (Mb-cyclic RGD) in UMI of arterial thrombi in vitro and in vivo . As expected, Mb-cyclic RGD had greater GP IIb/IIIa-targeted binding capability in all shear stress conditions. In addition, the shear stress at half-maximal detachment of Mb-cyclic RGD was 5.7-fold higher than that of microbubbles with non-specific peptide (Mb-CON) (p<0.05). Mb-cyclic RGD enhanced the echogenicity of the platelet-rich thrombus in vitro whereas Mb-CON did not produce enhancement. In the in vivo setting, optimal signal enhancement of the abdominal aortic thrombus was displayed with Mb-cyclic RGD in all cases. Mean video intensity of the abdominal aortic thrombi with Mb-cyclic RGD was 3.2-fold higher than that with Mb-CON (p<0.05). The novel Mb-cyclic RGD facilitated excellent visualisation of arterial thrombi using UMI and showed great promise for clinical applications.

Ultrasound molecular imaging of angiogenesis induced by mutant forms of hypoxia-inducible factor-1α.

AIMS: Targeted point mutants of hypoxia-inducible factor-1α (HIF-1α) are potential optimal agents for angiogenesis therapy. Data are limited regarding the angiogenic response of HIF-1α mutants. We aimed to compare the angiogenic effect of wild-type and mutant HIF-1α by contrast ultrasound molecular imaging (UMI) of α(v)-integrin expression. METHODS AND RESULTS: The wild-type gene of human HIF-1α, a gene with double mutations (HIF-1α(564/803)), a gene with triple mutations (HIF-1α(564/803/402)), or the LacZ gene (control) was transfected into the ischaemic hind limbs of C57BL/6 mice using an adenovirus vector. The video intensity of microbubbles targeted to α(v)-integrins in the ischaemic limbs increased along with the number of point mutations of HIF-1α. Immunohistochemical expression of endothelial α(v)-integrins was higher in the mutant HIF-1α(564/803/402) group than the other groups as was the density of both capillaries and arterioles in ischaemic muscle. Expression of both the mRNA and protein for HIF-1α and VEGF was significantly higher in the mutant HIF-1α(564/803/402) group than in the other groups. The half-life of HIF-1α and VEGF mRNA was longer in HIF-1α mutant-transfected cells than in wild-type HIF-1α or LacZ-transfected cells. CONCLUSION: HIF-1α mutants were more effective for enhancing angiogenesis in ischaemic muscle tissue than wild-type HIF-1α, and the response could be qualitatively evaluated by UMI of α(v)-integrins expression.

Late-phase detection of recent myocardial ischaemia using ultrasound molecular imaging targeted to intercellular adhesion molecule-1.

AIMS: in this study, we attempted to detect a recent myocardial ischaemic event using ultrasound molecular imaging (UMI) with microbubbles (MB) targeted to intercellular adhesion molecule-1 (ICAM-1) in the late phase of reperfusion. METHODS AND RESULTS: we created a myocardial ischaemia-reperfusion model in 60 C57/BL male mice to simulate an angina attack (ischaemia for 15 min, reperfusion for 1-24 h). The degree of myocardial inflammation and levels of ICAM-1 protein were determined by histological and immunohistochemical analyses. UMI with MB targeted to endothelial ICAM-1, as well as routine non-invasive methods including electrocardiography, echocardiography, and plasma troponin I levels, were utilized to evaluate ischaemia over the time course of reperfusion. Levels of ICAM-1 in the vascular endothelium were significantly increased over the time course of reperfusion (8-24 h) of the ischaemic myocardium. The video intensity of ICAM-1 molecular images of the ischaemic anterior wall was almost three times greater than that in the non-ischaemic posterior wall during the late phase (8-24 h) of reperfusion. In contrast, routine methods yielded only weak evidence of ischaemia. CONCLUSION: UMI with MB targeted to endothelial ICAM-1 provides reliable evidence of a recent myocardial ischaemic event in the late phase of reperfusion.

[Evaluation of tumor angiogenesis using microbubbles conjugated with RGD peptides and contrast enhanced ultrasound].

OBJECTIVE: To assess the feasibility of usage of microbubbles conjugated with RGD peptides and contrast enhanced ultrasound (CEU) in detection of tumor angiogenesis. METHODS: Lipid microbubbles (MB) were prepared, and the RGD peptides were covalently conjugated to the lipid shell of MB (MB(RGD)). Six nude mice with tumor created by dorsal inoculation of HepG2 tumor cells were used as the test group. Six nude mice without tumor were served as the control group. 10 minutes after bolus injection of MB and MB(RGD) randomly (30 min interval) via a tail vein catheter, CEU was performed on the tumors of the test group and the thigh skeletal muscles of control group. The video intensity (VI) of tumors and the skeletal muscles were measured. The tumors and the skeletal muscles were harvested for immunohistochemical examination. RESULTS: Only a slight contrast enhancement of the tumor was seen with MB, and the VI was 5.33 ± 1.71. While a remarkable enhancement of the tumor was observed after injection of MB(RGD). The VI was up to 17.03 ± 3.58, 3.18 folds higher as compared with that obtained by injection of MB (P < 0.05). As expected, there were no obvious contrast enhancement of the skeletal muscles with both MB(RGD) and MB. There was a high expression of αvß3-integrin in tumor neovascular endothelium, however, no apparent expression of αvß3-integrin was observed in the skeletal muscle vascular endothelium. CONCLUSION: CEU with MB(RGD) can be used to effectively evaluate the angiogenesis of tumors, and it may greatly contribute to the early judgement of the nature of tumor.