Synopsis

Abdominal aortic aneurysms (AAA) are a relatively common disease, with still unclear etiology that is associated with high mortality due to aortic rupture. Here we show that mice deficient for the Mas-receptor show aggravated AAA formation upon Ang-II treatment and that accumulation of macrophages in bulk aneurysms can be non-invasively visualized by ¹H/¹⁹F MRI using intravenously applied perfluorocarbon nanoemulsions which are efficiently phagocytosed by macrophages. We conclude that Mas-receptor deficiency leads to increased inflammation with enhanced AAA formation and that ¹⁹F MRI-based inflammation imaging will help to further unravel the role of monocytes/macrophages in the course of AAA progression.

Background

Abdominal aortic aneurysms (AAA) are a relatively common disease which is associated with high mortality due to aortic rupture (1). The precise biological reasons for the formation and progression of aneurysms are still unclear, but there is evidence that hypertension induced by the renin-angiotensin system as well as inflammatory processes are important factors. Recently, it has been recognized that the angiotensin/Mas receptor pathway is a major counterbalancing mechanism of the renin-angiotensin system and that effects triggered by the Mas receptor (MasR) are crucial to attenuate the inflammatory and remodeling processes in different pathophysiological conditions.

Several imaging modalities are utilized to diagnose and monitor AAAs, however imaging the inflammatory component of AAAs is still difficult. Our group has recently utilized intravenously injected perfluorocarbon nanoemulsions (PFC) - which are phagocytosed by monocytes and macrophages - to unequivocally visualize inflammation in a variety of clinically relevant disease models by combined ¹H/¹⁹F MRI (2-5). Due to the absence of any ¹⁹F background in biological tissue, these ¹⁹F signals are highly specific and the combination with anatomical ¹H images enables a precise localization of inflammatory hot-spots. In the present study we aimed (i) to apply this approach for monitoring of monocyte/macrophage infiltration during development of AAAs and (ii) to investigate the consequences of a MasR depletion on AAA formation by ¹⁹F MRI-based inflammation imaging.

Methods

ApoE^-/- and ApoE^-/-/MasR^-/- mice (fed with normal diet) were treated with angiotensin II (1000 ng/kg/min) via subcutaneously implanted osmotic minipumps for 28 days and monitored longitudinally by ¹H/¹⁹F MRI at 9.4T using a microimaging unit with actively shielded 40-mm gradients (1.5 T/m maximum gradient strength, 110 µs rise time at 100 % gradient switching). Mice were placed in a 25-mm ¹H/¹⁹F birdcage resonator and after acquisition of anatomical ¹H reference images, MR angiography was performed using a flow compensated 2D flash sequence (256x192 matrix size; 0.5 mm slice thickness, 0.25 mm overlap; scan time 3 min; 80° flip angle).

To monitor macrophage infiltration, PFC were injected on day 4 and the ¹H/¹⁹F MRI datasets were acquired 72 h later, to ensure optimal accumulation of PFC-loaded cells at inflammatory foci. For ¹⁹F imaging, a ¹⁹F RARE sequence with the following parameters was applied: 2.56´2.56 cm2 FoV, 64×64 matrix, 1 mm slice thickness, TR 4000 ms, RARE factor 32, 256 averages, 34 min acquisition time. Ex vivo high resolution ¹H/¹⁹F MRI scans were performed using a 3D RARE sequence with the following parameter: 1.0´1.0´3.0 cm3 FoV, 128×128×192 matrix (¹⁹F = 64×64×96), TR 2500 ms, RARE factor 32, 8 averages (¹⁹F=180), 5 h (¹⁹F = 14 h) acquisition time.

Results and Discussion

₁H MR angiography clearly revealed that Ang-II-induced hypertension led an increased lumen diameter of abdominal aorta in the suprarenal area of both ApoE^-/- and ApoE^-/-/MasR^-/- mice within 28 days (Fig. 1A/B). However, this effect was more pronounced in ApoE^-/-/MasR^-/- compared to ApoE^-/- mice (Fig. 1B), although the arterial blood pressure was similar in both mouse strains (Fig. 1C). The increased volume load of the aorta was also associated with a more frequent appearance of bulk aneurysms in ApoE-/-/MasR-/- (~ 72%) compared to ApoE-/- mice (~ 40%) and could already be observed by ¹H MR angiography on day 7 of the Ang-II treatment.

To analyze the accompanying macrophage infiltration into the abdominal aorta, we injected PFCs on day 4 after Ang-II treatment and subjected mice 72 h afterwards to ¹⁹F MRI for efficient accumulation of PFC-loaded cells in inflammatory foci. Already at this early time, we found strong ¹⁹F signals surrounding the abdominal aorta in ApoE^-/-/MasR^-/-, however only in those areas with bulk aneurysms (Fig. 2A). The presence of ¹⁹F signals - which indicates macrophage infiltration into the injured vessel wall - was corroborated by high resolution ex vivo ¹H/¹⁹F MRI of isolated and fixed abdominal aortae (Fig. 3). Finally, histological analysis confirmed the presence of large amounts of macrophages within this area.

Conclusions

Acknowledgements

References

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