Original Article
In vivo molecular imaging of myocardial angiogenesis using the αvβ3 integrin-targeted tracer 99mTc-RAFT-RGD

https://doi.org/10.1007/s12350-010-9191-9Get rights and content

Abstract

Background

Myocardial angiogenesis following reperfusion of an infarcted area may impact on patient prognosis and pro-angiogenic treatments are currently evaluated. The non-invasive imaging of angiogenesis would therefore be of potential clinical relevance in these settings. 99mTc-RAFT-RGD is a novel 99mTc-labeled tracer that targets the αvβ3 integrin. Our objective was to determine whether this tracer was suitable for myocardial angiogenesis imaging.

Methods and Results

A rat model of reperfused myocardial infarction was employed. Fourteen days following reperfusion, the animals were injected with 99mTc-RAFT-RGD or with its negative control 99mTc-RAFT-RAD. Fourteen animals were dedicated to autoradiographic imaging, infarct staining, and gamma-well counting of myocardial activity. In vivo dual-isotope pinhole SPECT imaging of 201Tl and 99mTc-RAFT-RGD or 99mTc-RAFT-RAD was also performed in 11 additional animals. Neovessels were observed by immunostaining in the infarcted and peri-infarct areas. 99mTc-RAFT-RGD infarct-to-normal ratios by gamma-well counting and ex vivo imaging (2.5 ± 0.6 and 4.9 ± 0.9, respectively) were significantly higher than those of 99mTc-RAFT-RAD (1.7 ± 0.2 and 2.2 ± 0.4, respectively, P < .05). The infarcted area was readily visible in vivo by SPECT with 99mTc-RAFT-RGD but not with 99mTc-RAFT-RAD (infarct-to-normal zone activity ratio, 2.5 ± 0.6 and 1.7 ± 0.4, respectively, P < .05).

Conclusion

99mTc-RAFT-RGD allowed the experimental in vivo molecular imaging of myocardial angiogenesis.

Section snippets

Synthesis

RAFT(cyclo[-RGDfK-])4 (RAFT-RGD) and the negative control RAFT(cyclo[-RADfK-])4 (RAFT-RAD) were synthesized as previously described.23

Labeling

125I labeling was performed by adding 20 to 50 μg of RAFT-RGD or RAFT-RAD to 10 μL of chloramin-T at 5 mg/mL (Amersham Pharmacia, Meylan, France) and ~18.5 MBq of 125I. After 20 minutes, the oxidation was stopped using 40 μL of sodium pyrosulfite (4 mg/mL). The radiochemical purity (RCP) was determined using thin-layer chromatography (TLC) using RP-18 TLC plates

In Vitro Experiments

The results are presented in Figure 1. There was a significant 3.3-fold higher radiolabeled RAFT-RGD uptake on HMVECs when compared to that of RAFT-RAD at all concentrations tested (Figure 1a). In addition, competition experiments indicated that radiolabeled RAFT-RGD uptake was inhibited by 60% in the presence of a 200-fold excess of unlabeled cRGD (Figure 1b).

CD31, αVβ3, and KiM2R Immunostaining

Results from CD31, αVβ3, and KiM2R immunostaining are presented in Figure 2. Endothelial CD31 expression was observed in the normal as

Discussion

The clinical importance of non-invasive angiogenesis assessment was recently emphasized in a pilot imaging study on patients.26 In the present study, experimental myocardial ischemia resulted in the induction of angiogenesis in the infarcted and peri-infarct zones at day 14 following reperfusion as previously described by others27 and as demonstrated by positive CD31 and αVβ3 immunostaining in the infarcted and peri-infarct zones. In addition to neovessels, macrophages are also known to express

Funding

Financial support was provided by the National Institute for Health and Medical Research (INSERM), the National Agency for Research and technology (ANRT), and ERAS Labo. The authors have no conflict of interest to disclose.

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