Elsevier

Nuclear Medicine and Biology

Volume 41, Issue 10, November–December 2014, Pages 785-792
Nuclear Medicine and Biology

Inflammation imaging of atherosclerosis in Apo-E-deficient mice using a 99mTc-labeled dual-domain cytokine ligand

https://doi.org/10.1016/j.nucmedbio.2014.08.004Get rights and content

Abstract

Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) play a critical role in initiating and accelerating atherosclerosis. This study evaluated the imaging properties of 99mTc-TNFR2-Fc-IL-1RA (99mTc-TFI), a dual-domain cytokine radioligand that targets TNF-α and IL-1β pathways, in assessing atherosclerosis development in apolipoprotein-E-deficient (ApoE/) mice.

Methods

The feasibility and specificity of detecting atherosclerosis with 99mTc-TFI SPECT imaging were investigated in ApoE/ and ApoE+/+ mice. Fifty-four ApoE/ mice were fed either an atherogenic diet (AGD) or a normal diet (ND) beginning at 5 weeks of age. Eighteen Apo-E wild-type (ApoE+/+) mice were fed an ND. Two groups of ApoE/ mice (n = 12 each group) on AGD and ND were imaged three times with 99mTc-TFI and a high-resolution SPECT system at 20–25, 30–40, and 48–52 weeks to study the evolution of atherosclerotic plaque.

Results

Focal radioactive accumulations in the aortic arch region were observed in the ApoE/ mice (n = 12) on AGD but not in the ApoE+/+ mice on ND (n = 10). Apo-E/ mice on ND (n = 11) exhibited lower radioactive uptake than ApoE/ mice on AGD (P < 0.05). Co-injection of an excess of cold ligand with 99mTc-TFI resulted in significant reduction of 99mTc-TFI uptake in the ApoE/ mice on AGD. Longitudinal studies showed that 99mTc-TFI uptake in the aortas of ApoE/ mice progressively increased from 20 to 48 weeks. Real-time PCR assays demonstrated that atherosclerotic aortas expressed significantly higher IL-1β and TNF-α than the aortas from wild-type controls.

Conclusions

Atherosclerotic plaques were detected by 99mTc-TFI imaging in ApoE/ mice. 99mTc-TFI is promising for specific detection of inflammatory response in atherosclerotic plaques.

Introduction

Atherosclerosis is a chronic inflammatory vascular disease that is mediated by a complex network of pro-inflammatory cytokines, including members of the interleukin family, tumor necrosis factor (TNF), and interferons (IFN) [1]. Molecular imaging of atherosclerotic plaque inflammation may provide an opportunity to measure the inflammatory molecular or cellular components in the plaques and characterize plaque progression and vulnerability [2]. However, imaging of plaque inflammatory components is challenging. The plaques are small, requiring high-spatial-resolution imaging devices for localization. The signal strength of reporter molecules may be weak, necessitating sensitive quantitative techniques for detection. An imaging agent must be able to bind specifically to inflammatory components with high affinity and adequate residence time for imaging. The imaging agent should be rapidly cleared from the circulation to provide sufficient contrast between inflammatory lesions and normal surrounding blood pool and tissues.

In view of the biological importance of interleukin-1β (IL-1β) and TNF-α in the development of atherosclerosis [3], [4], imaging of IL-1β and TNF-α activities may be a sensitive indicator of plaque inflammatory reactions and response to therapeutic interventions. IL-1β and TNF-α have coordinated effects in mediating inflammatory reactions [5], [6], [7]. A dual-domain fusion protein, TNFR2-Fc-IL-1RA (TFI), has been developed in recent years for functional targeting of IL-1β and TNF-α pathways [8]. TFI consists of a TNF-neutralizing domain that specifically binds to TNF-α, and an IL-1 receptor antagonist domain. We have previously demonstrated that 99mTc-labeled TFI (99mTc-TFI) can target inflammatory sites with more potent affinity and increased radioactive uptake compared to the individual cytokine radioligands, 99mTc-TNFR2-Fc and 99mTc-IL-1RA-Fc [9], [10]. We hypothesized in this study that 99mTc-TFI imaging might permit specific assessment of inflammatory reactions in atherosclerotic plaques. To test our hypothesis and determine potential clinical usefulness of 99mTc-TFI, we performed SPECT imaging studies with 99mTc-TFI in apolipoprotein-E-deficient (ApoE/) mice using multiple experimental protocols. We investigated whether interrogation of the inflammatory response via IL-1β and TNF-α pathways could provide monitoring of atherosclerotic plaque evolution.

Section snippets

Radiolabeling and cell binding

TFI was obtained from AmProtein Corporation (San Gabriel, CA). The molecular size of TFI as determined by SDS-PAGE is 84 kDa under reducing conditions and 168 kDa under non-reducing conditions [8]. TFI was 99mTc-labeled using a modified indirect labeling protocol with succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic) (Solulink, San Diego, CA) as a chelator [11]. The protein was chelated with S-HyNic at a molar ratio 1:6. HyNic-conjugated TFI (0.1 mg) was mixed with 925 MBq 99mTcO4 (0.5

Radiolabeling and cell binding

The radiolabeling yield of 99mTc-TFI was typically greater than 80%. After gel purification, 99mTc-TFI RCP was always greater than 98% for animal injection. Binding of 99mTc-TFI to leukocytes gradually decreased with addition of increasing amounts of non-radiolabeled TFI, as shown in Fig. 1. The IC50 for 99mTc-TFI was 48.04 nM (n = 3).

Feasibility of detecting atherosclerosis in vivo

In 48–52-week-old ApoE/ mice on AGD, focal radioactive accumulation (hot spot) in the aortic arch region became visible around 60 min and remained detectable over

Discussion

Cytokine-based SPECT imaging could be a specific measure of plaque development linked directly to inflammatory response. Previously, 99mTc-labeled interleukin-2 (IL-2) uptake in carotid plaques was used to measure absolute number of lymphocytes expressing IL-2 receptors in the plaque, suggesting that non-invasive cytokine imaging can allow quantification of atherosclerotic plaque inflammation [13]. 99mTc-TFI imaging data in this study provide implications for clinical detection of

Conclusion

Atherosclerotic plaques in ApoE/ mice were noninvasively detected by high-resolution SPECT imaging using 99mTc-TFI. This dual-domain cytokine radioligand showed increasing uptake during plaque evolution, as confirmed by histological examinations and cytokine measurements. Bifunctional imaging with 99mTc-TFI may exploit the synergy between IL-1β and TNF-α pathways and may enable characterization of vascular plaques undergoing inflammation at different phases. Further studies are warranted to

Acknowledgments

The authors wish to thank Dr. Gail Stevenson for support in animal studies. We thank Edward Abril from the University of Arizona Tissue Acquisition and Cellular/Molecular Analysis Shared Service, which is supported by the Arizona Cancer Center Grant (NIH CA023074), for generating the histological data. This work was supported by NIH grants NHLBI R01-HL090716 and NIBIB P41-EB002035. Dr. Mizhou Hui is Chief Scientific Officer of AmProtein Corporation.

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