Original Article
Cell proliferation detected using [18F]FLT PET/CT as an early marker of abdominal aortic aneurysm

https://doi.org/10.1007/s12350-019-01946-yGet rights and content

Abstract

Background

Abdominal aortic aneurysm (AAA) is a focal aortic dilatation progressing towards rupture. Non-invasive AAA-associated cell proliferation biomarkers are not yet established. We investigated the feasibility of the cell proliferation radiotracer, fluorine-18-fluorothymidine ([18F]FLT) with positron emission tomography/computed tomography (PET/CT) in a progressive pre-clinical AAA model (angiotensin II, AngII infusion).

Methods and Results

Fourteen-week-old apolipoprotein E-knockout (ApoE−/−) mice received saline or AngII via osmotic mini-pumps for 14 (n = 7 and 5, respectively) or 28 (n = 3 and 4, respectively) days and underwent 90-minute dynamic [18F]FLT PET/CT. Organs were harvested from independent cohorts for gamma counting, ultrasound scanning, and western blotting. [18F]FLT uptake was significantly greater in 14- (n = 5) and 28-day (n = 3) AAA than in saline control aortae (n = 5) (P < 0.001), which reduced between days 14 and 28. Whole-organ gamma counting confirmed greater [18F]FLT uptake in 14-day AAA (n = 9) compared to saline-infused aortae (n = 4) (P < 0.05), correlating positively with aortic volume (r = 0.71, P < 0.01). Fourteen-day AAA tissue showed increased expression of thymidine kinase-1, equilibrative nucleoside transporter (ENT)-1, ENT-2, concentrative nucleoside transporter (CNT)-1, and CNT-3 than 28-day AAA and saline control tissues (n = 3 each) (all P < 0.001).

Conclusions

[18F]FLT uptake is increased during the active growth phase of the AAA model compared to saline control mice and late-stage AAA.

Introduction

Abdominal aortic aneurysm (AAA) is a focal dilatation of the abdominal aorta that progresses to rupture, which confers high mortality.1 AAA is detected incidentally or through ultrasound scanning (USS)-based screening programmes.2,3 Once detected, USS monitoring is used to track AAA diameter until it reaches the 5.5-cm intervention threshold; this can take > 10 years and places a discernible burden on patients.4,5 A non-invasive stratification biomarker applied at the time of AAA detection to personalize monitoring regimens and identify those who may benefit from a specific medical therapy or early intervention would be beneficial. Positron emission tomography (PET) can provide information about the molecular processes beyond the anatomical characteristics of USS or computed tomography (CT) and may therefore be useful for this purpose.

Fluorine-18 fluorodeoxyglucose ([18F]FDG) is a glucose uptake marker commonly used in oncology as an indicator of high metabolic activity, such as at sites of inflammation, and is the most commonly administered PET radiotracer in clinical practice. The data on [18F]FDG uptake in AAA are mixed and conflicting.6, 7, 8, 9 Some studies suggest that increased [18F]FDG uptake positively correlates with AAA progression,10, 11, 12, while others indicate an inverse relationship or lack of correlation between [18F]FDG uptake and aortic size.13,14 The Sodium Fluoride Imaging of AAA (SoFIA3) trial recently demonstrated that fluorine-18 sodium fluoride AAA uptake (reflecting microcalcification) predicts AAA progression and rupture, providing the first proof-of-concept data for PET/CT as a AAA stratification biomarker 15; however, the mechanism remains unclear.

Maegdefessel et al. showed that modulating microRNA-21 expression reduces cell proliferation, protecting mice from AAA expansion.16 Smooth muscle cell de-differentiation and proliferation is an early event in AAA.17 Preventing this event by deleting Kruppel-like factor-4 prevents AAA formation in mice.18 These data point to a period of pathological cellular remodeling in early AAA and suggest that anti-proliferative therapy might be beneficial. Therefore, detecting cell proliferation in vivo may be useful. Our hypothesis is that this proliferative remodeling is detectable using PET/CT with an analogue of the pyrimidine deoxynucleoside thymidine, fluorine-18 fluorothymidine ([18F]FLT). This tracer accumulates in proliferating cells and reflects thymidine kinase-1 (TK-1) activity. Here, we investigate [18F]FLT as a PET/CT radiotracer in the progressive angiotensin II (AngII) infusion pre-clinical model of AAA.

Section snippets

Animals

All animal work was conducted in accordance with the UK Home Office, Animals (Scientific Procedures) Act 1986 under Project Licence P606320FB. Male Jax™ apolipoprotein E-knockout mice (ApoE−/−; B6.129P2-Apoetm1Unc/J; Charles River, UK) were used for experiments at 14 weeks of age. Male C57BL6/J mice (Charles River, UK) were used at 8 weeks of age for baseline biodistribution studies. All animals were part of the Jackson Laboratories Genetic Stability Programme to limit cumulative genetic drift.

Cell Proliferation Occurs in AngII AAA

As expected, ApoE−/− mice infused with AngII developed significant supra-renal aortic lumen dilatation compared to the saline-infused controls (Figure 1A). To test for cell proliferation in AngII AAA, we stained fixed supra-renal abdominal aortic tissue from ApoE−/− mice infused with saline or AngII for 28 days with Ki67, which accumulates in cells throughout the S, G2, and M cell cycle phases.21 A significant increase in the proportion of Ki67-positive nuclei was observed in the aortic wall in

Discussion

This is the first study to explore the feasibility of [18F]FLT PET/CT in an experimental murine model of progressive AAA. We demonstrated that [18F]FLT uptake in AngII-induced AAA increased after 14 days of infusion compared to saline controls with subsequent signal fall-off after 28 days. This was corroborated by a similar pattern of TK-1 protein expression in an independent cohort of animals. We confirmed that the [18F]FLT signal was originating from the aortic tissue by performing ex vivo

New Knowledge Gained

In 14-day AngII-induced AAA compared to 28-day AngII-induced AAA and saline controls, the [18F]FLT PET signal is significantly enhanced and [18F]FLT counts are greater, which themselves correlate with aortic volume. The expression of the [18F]FLT substrate, TK-1, and [18F]FLT transporter proteins ENT-1, ENT-2, CNT-1, and CNT-3 are also increased in AngII AAA. These findings suggest an early period of cell proliferation in the AngII AAA murine model, which is detectable using PET/CT.

Conclusions

[18F]FLT is increased during the active growth phase of the AngII AAA murine model compared to saline control animals or late-stage AngII AAA. [18F]FLT uptake is correlated with aortic volume. The expression of the [18F]FLT substrate TK-1 is also increased in the model. Further work is necessary to determine the cell types contributing to the proliferative signal.

Acknowledgements

We thank Melanie Reay, Laura Goodlass, and Danielle Webster for their technical work maintaining the animals during the study.

Author Contributions

RG—performed experiments and collected data, analyzed data from experiments, wrote manuscript, obtained funding. CC—performed experiments and collected data, edited manuscript. LC—performed experiments and collected data, edited manuscript. JW—performed experiments and collected data, edited manuscript. JD—produced critical reagents for experiments. PH—produced critical

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    Increased cellular proliferation in the AngII abdominal aortic aneurysm mouse model can be detected using [18F]FLT PET/CT.

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