J Cardiovasc Ultrasound.  2010 Sep;18(3):91-97. 10.4250/jcu.2010.18.3.91.

Targeted Ultrasound Imaging of Apoptosis with Annexin A5 Microbubbles in Acute Doxorubicin-Induced Cardiotoxicity

• Min PK
• Lim S
• Kang SJ
• Hong SY
• Hwang KC
• Chung KH
• Shim CY
• Rim SJ
• Chung N

• KMID: 2177306
• DOI: http://doi.org/10.4250/jcu.2010.18.3.91

Abstract

BACKGROUND
The aim of this study was to assess the feasibility of targeted ultrasound imaging on apoptosis with annexin A5 microbubbles (A5MB) in acute doxorubicin-induced cardiotoxicity.
METHODS
Avidinated and octafluoropropan-filled phospholipid microbubbles were conjugated with biotinylated annexin A5. To confirm the specific binding of A5MB, flow cytometry was performed with hydrogen peroxide induced apoptosis in rat aorta smooth muscle cells incubated with fluorescein-5-isothiocyanate (FITC) labeled annexin A5 and A5MB. Adult male rats were injected intraperitoneally with 5 mg/kg doxorubicin weekly for 3 weeks (n = 5). Control rats were injected with normal saline (n = 5). At 24 hours after the final treatment, triggering imaging was performed 15 min after an intravenous bolus injection of A5MB for washout of freely circulating microbubbles. After echocardiography, the heart was isolated for histological detection of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay.
RESULTS
In the in vitro tests, fluorescence intensity was low for healthy cells and high for apoptotic cells when incubated with FITC-labeled annexin A5 and A5MB. Rats treated with doxorubicin showed significant contrast opacification of the myocardium on contrast echocardiography using A5MB. However, no opacification was observed in control rats. Apoptosis was confirmed by TUNEL assay in doxorubicin treated rats.
CONCLUSION
Acute doxorubicin-induced cardiomyopathy based on early apoptosis can be assessed and imaged with targeted ultrasound imaging using A5MB in rats.

Keyword

Cardiomyopathy; Apoptosis; Annexin A5; Microbubbles; Echocardiography

MeSH Terms

Adult
Animals
Annexin A5
Aorta
Apoptosis
Avidin
Cardiomyopathies
Doxorubicin
Echocardiography
Flow Cytometry
Fluorescein-5-isothiocyanate
Fluorescence
Heart
Humans
Hydrogen Peroxide
In Situ Nick-End Labeling
Male
Microbubbles
Myocardium
Myocytes, Smooth Muscle
Rats
Annexin A5
Avidin
Doxorubicin
Fluorescein-5-isothiocyanate
Hydrogen Peroxide

Figure

• 

  Fig. 1 Specific binding of FITC-labeled annexin A5 to apoptosis cells. Healthy cells (A). Healthy cells incubated with FITC-labeled annexin A5 (B). Healthy cells incubated with microbubbles conjugated with FITC-labeled annexin A5 (C). Apoptotic cells incubated with FITC-labeled annexin A5 (D). Apoptotic cells incubated with microbubbles conjugated with FITC-labeled annexin A5 (E). FITC: fluorescein-5-isothiocyanate.

• 

  Fig. 2 Targeted ultrasound imaging with annexin A5 conjugated microbubbles in doxorubicin-treated rats (A and B) and control rats (C and D). Significant contrast opacification of myocardium (arrows) was observed in the 1st frame (A). Initial contrast enhancement was dissipated during subsequent high mechanical index imaging in the 10th frame (B). No contrast opacification was observed in the myocardium in the 1st frame (C). No contrast opacification was observed in the myocardium in the 10th frame (D).

• 

  Fig. 3 In situ labeling of DNA fragmentation in rat myocardium from TUNEL assay. Fragmented DNA is labeled in brown, and all nuclei are counterstained with methyl-green. Myocardial sections from rats in the control group reveal no evidence of DNA fragmentation in cardiomyocytes (A) or endothelial cells (C). A heterogenous pattern is revealed for the isolated apoptotic nuclei in the myocardium of doxorubicin-treated rats, as indicated by arrows, for both cardiomyocytes (B) and endothelial cells (D). TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

• 

  Fig. 4 The quantitative results of positive TUNEL-staining cardiomyocytes and endothelial cells. The ratio of TUNEL-positive cardiomyocytes (A) and endothelial cells (B) are significantly higher in the doxorubicin-treated group (Doxo) than in the control group (Con). *p < 0.01 vs. control. TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

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