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. Author manuscript; available in PMC: 2016 Jan 30.
Published in final edited form as: Circ Cardiovasc Imaging. 2014 Dec 30;8(1):10.1161/CIRCIMAGING.114.002471 e002471. doi: 10.1161/CIRCIMAGING.114.002471

Imaging vessel wall biology to predict outcome in abdominal aortic aneurysm

Reza Golestani 1,2,*, Mahmoud Razavian 1,2,*, Lei Nie 1,2, Jiasheng Zhang 1,2, Jae-Joon Jung 1,2, Yunpeng Ye 1,2, Michelle de Roo 1,2, Koen Hilgerink 1,2, Chi Liu 3, Simon P Robinson 4, Mehran M Sadeghi 1,2
PMCID: PMC4284949  NIHMSID: NIHMS645652  PMID: 25550400

Abstract

Background

Abdominal aortic aneurysm (AAA) rupture risk is currently determined based on size and symptoms. This approach does not address the rupture risk associated with small aneurysms. Given the role of matrix metalloproteinases (MMPs) in AAA weakening and rupture, we investigated the potential of MMP-targeted imaging for detection of aneurysm biology and prediction of outcome in a mouse model of AAA with spontaneous rupture.

Methods and Results

Fifteen week-old mice (n=66) were infused with angiotensin II for four weeks to induce AAA. Saline-infused mice (n=16) served as control. The surviving animals underwent in vivo MMP-targeted microSPECT/CT imaging, using RP805, a 99mTc-labeled MMP-specific tracer, followed by ex vivo planar imaging, morphometry and gene expression analysis. RP805 uptake in suprarenal aorta on microSPECT images was significantly higher in animals with AAA, as compared with angiotensin II-infused animals without AAA or control animals. CD68 expression and MMP activity were increased in AAA and significant correlations were noted between RP805 uptake and CD68 expression or MMP activity, but not aortic diameter. A group of angiotensin II-infused animals (n=24) were imaged at 1 week and were followed for an additional three weeks. RP805 uptake in suprarenal aorta at 1 week was significantly higher in mice that later developed rupture/AAA. Furthermore, tracer uptake at 1 week correlated with aortic diameter at 4 weeks.

Conclusions

MMP-targeted imaging reflects vessel wall inflammation and can predict future aortic expansion or rupture in murine AAA. If confirmed in humans, this may provide a new paradigm for AAA risk stratification.

Keywords: Aneurysm, Inflammation, Imaging, Remodeling

Introduction

Vessel wall inflammation and matrix remodeling accompany the development, expansion, and rupture of abdominal aortic aneurysm (AAA) 1, 2. While the causal role of inflammation in the development of AAA can be debated, the association of vessel wall inflammation and matrix remodeling with changes in tensile strength, aortic expansion, and aortic rupture is well-documented 2. The recruitment and proliferation of leukocytes within the aortic wall and release of mediators which promote matrix metalloproteinase (MMP) expression and activation are key determinants of clinical outcome in AAA 1, 2. However, in the current management guidelines these and other key molecular and cellular processes in the pathogenesis of AAA are neglected and clinical decisions are made solely based on aneurysm size, expansion rate, and/or symptoms 3, 4. As such, the rupture risk associated with small AAAs is not addressed by current guidelines, and the management strategies may impose an unnecessary risk of repair surgery to subjects with large but stable AAA.

Molecular imaging aimed at key pathological processes involved in aneurysm growth and rupture can provide unique information to complement current AAA management strategies. Somewhat based on the clinical availability of 18F-fluorodeoxyglucose (FDG) and its application in imaging inflammation in other organs, 18F-FDG positron emission tomography (PET) has been proposed for detection of vessel wall inflammation and determining aortic aneurysm propensity to expansion and rupture 5–7. While 18F-FDG uptake has been shown to relate to leukocyte infiltration within the vessel wall 8, 9, there is ongoing debate on determinants of vascular 18F-FDG signal 10–12. Accordingly, a recent study to evaluate the predictive value of 18F-FDG PET imaging in AAA showed an inverse relation between 18F-FDG uptake and future expansion or rupture in subjects with AAA 6, highlighting the necessity of more specific tracers for imaging inflammation and remodeling in AAA.

MMPs play a key role in vascular remodeling 2, 13, 14. Radiolabeled tracers which bind to activated MMPs have shown promise for imaging vascular remodeling by micro single photon emission tomography (SPECT) imaging 15, 16. In a previous study we showed that MMP-targeted imaging can predict arterial expansion in a surgical model of carotid artery aneurysm 16. In that study, MMP signal was specific, and paralleled CD68 expression in the vessel wall. While promising, the surgical nature of carotid aneurysm which prevented the unequivocal discrimination of tracer uptake in the vessel wall from uptake in the surrounding surgical wound, and the absence of spontaneous aneurysm rupture which prevented assessing the risk of rupture mandated further evaluation in clinically relevant animal models. As a bridge to future clinical studies, we investigated the feasibility of MMP-targeted imaging for detection of MMP activity and inflammation in a non-surgical murine model of AAA with potential for spontaneous aortic rupture. Here, we demonstrate the feasibility and effectiveness of MMP imaging in AAA and establish its predictive value for AAA propensity to expansion and rupture.

Materials and Methods

Please refer to supplemental data for details of materials and methods.

Animal Model

An osmotic minipump (ALZET model 2004) was implanted subcutaneously in 13–15 weeks old male mice under isoflurane (1–3%) anesthesia, through which angiotensin II (1000μg/kg/day, n = 66, Supplemental table) was administered for up to 28 days. To achieve a broad range of responses, B6.129P2-Apoetm1Unc/J and C57BL/6J mice from Jackson Laboratories and their offspring were used for this study. Animals infused with normal saline or nothing (n=16) were used as control. Experiments were performed according to regulations of Yale University and VA Connecticut Healthcare System’s Animal Care and Use Committees.

Imaging

The precursor form of RP805, a 99mTc-labeled tracer with specificity for activated form of MMPs was radiolabeled as described17. MicroSPECT/CT imaging was performed on a high-resolution small animal imaging system (X-SPECT, Gamma Medica-Ideas, Northridge, CA) equipped with 1-mm low-energy pinhole collimators, as described with minor modifications15, 16, 18.

Statistical analysis

All data are presented as mean ± SD. Levene’s test was used to assess equality of variances. The statistical significance of the data in multiple groups with equal variances was examined using one-way ANOVA with Tukey’s post-hoc analysis. If Levene’s test indicated that variances were not equal, the Welch’s ANOVA with Games-Howell post hoc procedure was used. Pearson’s correlation coefficient for linear regression was calculated to test the association between different parameters. Welch t-test was used to compare tracer uptake at 1 week between those mouse that developed AAA or rupture and those without AAA at 4 weeks. Statistical significance was set at p<0.05.

Statistical analysis was performed using GraphPad Prism version 6.03 for Windows or SPSS Statistics for Windows, Version 21.0 (for Levene’s test and Welch’s ANOVA).

Results

Of 66 mice infused with angiotensin II for up to four weeks, 17 animals (26%) died due to aortic rupture ascertained thorough post-mortem examination. In contrast, all saline-infused, control mice survived during this period (Figure 1A). On ex vivo morophometric analysis at 4 weeks, the maximal external diameter of suprarenal abdominal aorta in the control group was 0.83 ± 0.07 mm. Based on this, the upper limit of normal diameter range was established at the mean ± 2 SD = 0.97 mm. Aortae above this upper limit were considered aneurysmal. With this definition, of those animals in the angiotensin II-infusion group that survived without rupture to 4 weeks and for whom the tissue was available, 36 had developed AAA. The maximal external aortic diameters in animals with and without AAA were, respectively, 1.62 ± 0.50, and 0.84 ± 0.10 mm, p < 0.0001 (Table 1, and Figure 1B and C, Supplemental Figure 1).

Figure 1.

Figure 1

Figure 1

Figure 1

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AAA development. A) Kaplan-Meier survival curves of mice infused with saline or angiotensin II for 4 weeks. B) Maximum external diameter of suprarenal aorta at 4 weeks in saline- or angiotensin II-infused mice. The dotted line represents the threshold (mean+2SD of maximum external aortic diameter of saline-infused, control mice, 0.97 mm) used to define AAA. C) Examples of Movat’s pentachrome staining of suprarenal aorta in a control, saline-infused mouse (left) and an angiotensin II-infused mouse with AAA (right). Scale bar: 100 μm. AngII: angiotensin II.

Table 1.

Overview of animals’ fate at 4 weeks. AngII: angiotensin II.

Group n AAA-related death (%) AAA (%) External diameter (mm)
AAA no AAA
Saline-infused 16 0 0 - 0.83 ± 0.07*
AngII-infused 66 17 (26%) 36 (55%) 1.62 ± 0.50 0.84 ± 0.10*
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*

: p < 0.0001 compared with AAA.

In vivo imaging of MMP activation in AAA

To detect and quantify MMP activation in AAA in vivo, a group of mice underwent RP805 (a 99mTc-labeled tracer that specificity targets MMP activation 17) microSPECT/CT imaging at 4 weeks after angiotensin II or saline infusion. While there was no visible focal suprarenal aortic uptake of RP805 in saline-treated mice, in a subset of angiotensin II-infused animals, microSPECT images obtained 2 hours after RP805 administration showed considerable tracer uptake in suprarenal abdominal aorta, as located by CT angiography (Figure 2A–C). Quantification of RP805 signal showed significantly higher suprarenal abdominal aorta tracer uptake in angiotensin II-infused mice as compared with saline-infused animals (0.11 ± 0.08 vs 0.04 ± 0.00 cpv per MBq injected dose, p < 0.0001, n = 33 and 9 for angiotensin II- and saline-infused mice, respectively). Morphometric analysis of aortas harvested after imaging confirmed the presence of AAA in 21 out of 33 angiotensin II-infused animals. When stratified based on the presence of AAA, MMP tracer uptake was significantly higher in angiotensin II-infused animals with AAA compared with those without AAA [0.15 ± 0.09 (n = 21) vs 0.05 ± 0.02 (n = 12) cpv per MBq injected dose, p < 0.0001, Figure 2D). RP805 uptake in AAA was also significantly higher than uptake in aortae of saline-infused mice (p < 0.0001, Figure 2D).

Figure 2.

Figure 2

Figure 2

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In vivo MMP-targeted imaging. A–C) Examples of fused RP805 microSPECT/CT angiography images of a control, saline-infused mouse (A), an angiotensin II-infused mouse without abdominal aortic RP805 uptake (B), and an angiotensin II-infused mouse with abdominal aortic RP805 uptake (C). Arrows point to suprarenal abdominal aorta. D) MicroSPECT/CT-derived quantification of suprarenal abdominal aorta RP805 uptake. * :p <0.0001 [(animals with AAA vs. animals without AAA (noAAA)], †: p < 0.0001 (animals with AAA vs. control animals). T: transverse, C: coronal, S: sagittal. cpv: counts per voxel, AngII: angiotensin II.

Ex vivo imaging

To validate in vivo imaging results, a subset of animals underwent ex vivo planar imaging following in vivo microSPECT/CT imaging. Similar to in vivo data, a focal aortic signal was readily detectable on planar images in animals with AAA (Figure 3A). Quantification of tracer uptake in suprarenal aorta showed significantly higher RP805 uptake in angiotensin II-infused animals with AAA as compared with saline-infused animals [0.52 ± 0.45 (n = 12) vs 0.05 ± 0.02 (n = 7) cpp per MBq injected dose, p = 0.01, Figure 3B]. The difference in RP805 uptake between angiotensin-infused animals with and without AAA was at borderline significance (p = 0.06). Importantly, there was an excellent correlation between in vivo and ex vivo quantification of aortic RP805 uptake (r = 0.89, P < 0.0001, Figure 3C).

Figure 3.

Figure 3

Figure 3

Figure 3

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Ex vivo RP805 imaging. A) Examples of an ex vivo planar image (right), and corresponding photograph (left) of an excised aorta with AAA. Scale bar: 5 mm. B) Planar imaging-derived quantification of RP805 uptake in suprarenal aorta. †: p = 0.01 (vs. saline-infused animals), ‡: p < 0.05 (vs. saline-infused animals). C) Correlation between microSPECT-derived and planar imaging-derived quantification of RP805 uptake in suprarenal aorta (r = 0.89, p< 0.0001). Dotted lines represent 95% confidence interval for the linear regression line. cpp: counts per pixel. cpv: counts per voxel.

Tissue characterization

The changes in aortic wall composition associated with AAA development were investigated by immunostaining and quantitative RT-PCR. As expected, in both control and angiotensin II-infused animals, aortic lumen was surrounded by a layer of CD31 positive endothelial cells, and the media consisted mostly of α-actin positive vascular smooth muscle cells. In addition, a number of CD31 and in some cases, smooth muscle α-actin positive small blood vessels were present in the adventitia of aneurysmal aortic segments. Compared with aortas of control animals, a prominent CD68 (macrophage marker)-positive inflammatory infiltrate was present in AAA (Figure 4A). This was confirmed by quantitative RT-PCR, which showed significantly higher GAPDH-normalized CD68 expression in AAA relative to control aortas (p < 0.05, n= 4–13 in each group, Figure 4B). Similarly, the development of AAA was associated with upregulation of several members of MMP family, including MMP-2, -9 and -12 as detected by quantitative RT-PCR (p < 0.05, n= 4–13 in each group, Figure 4C). Finally, MMP activation assessed by zymography was significantly higher in AAA compared with saline-infused, control aortas (p < 0.05, n = 4–12 in each group, Figure 4D).

Figure 4.

Figure 4

Figure 4

Figure 4

Figure 4

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Tissue characterization. A) Examples of CD68 (top row), CD31 (middle row) and smooth muscle α-actin (bottom row) immunohistochemistry in aneurysmal (right) and control (left) aortae. Scale bar: 100 μm. B) Quantitative RT-PCR assessment of CD68 expression in abdominal aorta of saline infused, control mice and angiotensin II-infused mice with AAA or without AAA (noAAA). †: p < 0.05 (vs. saline-infused), n = 8, 4, 13, respectively for saline-infused, noAAA, and AAA. C) Quantitative RT-PCR assessment of MMP-2, -9 and -12 expression in abdominal aorta of saline infused, control mice and angiotensin II-infused mice with AAA or without AAA (noAAA). †: p < 0.05 for MMP-2 and MMP-12 and p < 0.01 for MMP-9 (vs. saline-infused animals), *: p < 0.05 (vs. noAAA). For MMP-2: n = 6, 4, 11, for MMP-9: n = 7, 4, 12; and for MMP-12: n = 7, 4, 12, respectively for saline-infused, noAAA, and AAA. D) Zymographic quantification of MMP activity in abdominal aorta of saline infused, control mice and angiotensin II-infused mice with AAA or without AAA (noAAA). †: p < 0.05 (vs. saline-infused animals), n = 7, 4, 9, respectively for saline-infused, noAAA, and AAA.

Correlates of MMP activation in AAA

To address the correlates of RP805 uptake in vivo aortic tissue was frozen immediately after microSPECT/CT imaging in a subset of animals (n = 15). Comparing microSPECT-derived MMP activity in abdominal aorta with aortic MMP activity assessed by zymography there was a significant and robust correlation between the two measures of MMP activity (r = 0.83, p < 0.001) (figure 5A). We also found a significant and robust correlation between microSPECT-derived MMP activity and CD68 expression (r = 0.89, p < 0.0001, Figure 5B). The correlation between RP805 uptake and zymography or CD68 expression persisted when only animals with AAA were analyzed (r = 0.79, p = 0.01 or 0.91, p < 0.01, respectively for zymography and CD68 expression, Supplemental Figure 2A and B). Next we investigated whether aortic size is a determinant of RP805 uptake. Combining both saline-infused and angiotensin II-infused animals, there was a modest, yet significant, correlation between RP805 uptake and aortic diameter (r = 0.47, p < 0.01) (figure 5C). However, in animals with AAA there was no correlation between RP805 uptake and aortic diameter (Supplemental Figure 2C), indicating that unlike MMP activity or CD68 expression, aortic diameter is not a determinant of RP805 uptake in AAA. Finally, there was a significant correlation between MMP activity quantified by zymography and CD68 expression (r = 0.74, p< 0.0001) (figure 5D).

Figure 5.

Figure 5

Figure 5

Figure 5

Figure 5

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Correlates of MMP activation in abdominal aorta. A–C) Correlation between RP805 uptake in suprarenal abdominal aorta and MMP activity by zymography (A), CD 68 expression by quantitative RT-PCR (B), and aortic diameter (C). D) Correlation between MMP activity and CD68 expression. Dotted lines represent 95% confidence interval for the linear regression line. AU: arbitrary unit.

Predictive value of RP805 imaging

Next we sought to investigate whether early MMP-targeted imaging can predict ultimate outcome in AAA. A group of mice (n = 24) underwent RP805 microSPECT imaging at one week after the initiation of angiotensin II infusion. These animals were allowed to recover from anesthesia, and were followed for three weeks after imaging. Similar to animals imaged at 4 weeks, RP805 signal was readily detectable in suprarenal aorta of a subset of animals imaged at 1 week (Figure 6). During the follow up observation period, as expected, a subset of these mice (n = 3) died of AAA rupture. Of the surviving animals, 12 had, and 9 did not have AAA on ex vivo morphometric analysis at 4 weeks. On retrospective analysis of images, mean tracer uptake in suprarenal abdominal aorta at one week in all angiotensin II-infused mice was 0.08 ± 0.05 cpv per MBq injected dose. RP805 at 1 week was significantly higher in animals who died of rupture or developed AAA than those who survived to 4 weeks without AAA (0.10 ± 0.05 vs. 0.03 ± 0.02 cpv per MBq injected dose, p < 0.0001) (Figure 6).Similarly, when analyzed as a continuous variable, there was a significant correlation between RP805 uptake at 1 week and aortic diameter at 4 weeks (R= 0.53, p < 0.01), indicating that early MMP-targeted imaging can predict the outcome in this model of AAA (Figure 6).

Figure 6.

Figure 6

Figure 6

Figure 6

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Predictive value of RP805 imaging. A) Examples of RP805 microSPECT/CT images at 1 week in an angiotensin II-infused mouse that did not (left) and a mouse that did (right) develop aneurysm at 4 weeks. Top row: transvers CT angiography images, Bottom row: transverse fused microSPECT and CT angiography images. Arrows point to suprarenal abdominal aorta. B) Quantification of microSPECT/CT-derived suprarenal aorta RP805 uptake in images obtained at one week in mice that developed AAA or died of AAA rupture by 4 weeks after starting angiotensin II infusion, and those that did not develop AAA, *: p= 0.001. C) Correlation between RP805 in suprarenal abdominal aorta at 1 week and aortic diameter at 4 weeks after angiotensin II infusion. Dotted lines represent 95% confidence interval for the linear regression line. cpv: counts per voxel.

Discussion

To our knowledge, this is the first study to evaluate MMP-targeted microSPECT/CT imaging for predicting outcome in AAA. In an animal model of AAA with potential for spontaneous rupture we demonstrate that RP805 uptake in abdominal aorta correlates well with vessel wall MMP activity and macrophage content. Importantly, we show that early SPECT imaging shows promise regarding the animals’ propensity for future aortic expansion or death from aneurysm rupture as a composite endpoint.

In the US, AAA is responsible for ~10,000 deaths per year, mostly due to rupture, 19. Clinically, AAA is often asymptomatic and the main goal of treatment is to avoid the development of morbid complications. This can be achieved through preventive measures, including surgical or endovascular aneurysm repair in those subjects who are at high risk for rupture. According to current management guidelines, AAA rupture risk is determined based on its size, retrospective expansion rate, or presence of symptoms. Thus, subjects with small asymptomatic AAA are managed conservatively using a watch-and-wait-strategy focused on treating risk factors and tracking aneurysm expansion 3. However, a notable number of aneurysm ruptures occurs during this period in small AAA which do not meet the criteria for invasive treatment 20. This calls for refining AAA risk stratification criteria based on state-of-the-art diagnostic tools and underlying pathobiology 21.

Molecular imaging techniques can be used to detect and quantify the abundance and activity of key molecular and cellular players in AAA development and rupture. Through release of pro-inflammatory chemokines and cytokines, and proteases such as MMPs, inflammatory cells contribute to aneurysm expansion and rupture 22, and thus could be suitable targets for molecular imaging of AAA 21. Indeed, it is shown that AAA expansion in humans is associated with a high density of inflammatory cells in the adventitia 23. The most advanced molecular imaging techniques for detection of vascular inflammation and phagocytic activity which have been used in both preclinical and clinical settings are 18F-FDG PET and iron oxide nanoparticle-based MRI 7, 24–26. 18F-FDG is not specific for inflammatory cells as it can be retained in any highly metabolizing cell, and 18F-FDG PET has shown contradicting results in predicting aortic aneurysm rupture 6, 9. Ultrasmall particles of iron oxide-based MRI has shown encouraging results in predicting AAA growth 26. However, a number of issues remain to be addressed regarding the utility of this approach, including interference from endogenous iron present due to hemorrhage or thrombus, the modest degree of signal loss, uncertainties about the quantification methodology, non-specificity for macrophages for example due to entrapment within thrombus, and non-targeted nature of USPIO particles 21, 27. Given the role of MMPs in the pathogenesis of aneurysm MMP-targeted imaging is an alternative approach for detection of vascular inflammation and remodeling in aortic aneurysm. Indeed, several members of MMP family are upregulated in ruptured human AAA and plasma levels of others are found to be elevated 13, 14. As such, MMPs seem to be suitable targets for molecular imaging of aneurysms to predict expansion and rupture. The MMP family of zinc-dependent endopeptidases consists of ~25 related proteins that are synthetized as proenzymes 28. Activation of MMP proenzymes (whether allosteric or proteolytic) exposes the catalytic domain targeted by 99mTc-RP805 and its 111In-labeled homologue, RP782, used to detect MMP activation in murine aneurysm 16. Other MMP-targeted probes, including an activatable fluorescent imaging agent, MMPsense 680, and a gadolinium-based MR contrast agent, P947, have been evaluated for imaging aneurysm in murine models 29–31. However, unlike MMP-specific radiotracers, it is not clear whether there is any potential path to application of these agents for non-invasive imaging in humans 21.

The murine model of AAA used in this study offers several advantages over other rodent models of AAA. In addition to the potential for spontaneous rupture, the angiotensin II-induced model of AAA recapitulates many aspects of human disease, including macrophage infiltration and MMP activation 32, 33, increasing the likelihood that the data obtained with this model are relevant to human AAA. Combining two strains of mice and their offspring which show different rates of aneurysm induction in our study 34 might have further increased this likelihood. The absence of a surgical wound nearby which can interfere with image analysis, as encountered in surgical models of AAA, is another practical advantage of angiotensin II-induced murine AAA. Indeed, target gene expression in the surrounding wound was a confounding factor in a previous study on imaging carotid aneurysm 16. While we have addressed MMP-specificity of tracer uptake in aneurysm using excess unlabeled precursor 16, the potential contribution of surgical wound and damage to perivascular tissue to tracer uptake in vivo remained a concern. Despite the absence of perivascular uptake of the tracer in our model, the small size of aorta in the mouse can be a challenge to in vivo quantification of tracer uptake. To address this concern, we validated in vivo microSPECT-derived quantitative measures of RP805 uptake using the data derived from ex vivo planar images of the corresponding segments of abdominal aorta, demonstrating an excellent correlation between in vivo and ex vivo measurements of tracer uptake.

Various groups of investigators have used different criteria to define AAA in the mouse 35–37. Here, to ascertain the validity of our results we used a rather stringent threshold based on mean + 2SD of aortic diameter in control mice to define AAA. We found MMP signal to be significantly higher in AAA than in abdominal aorta of saline-treated, control mice or those angiotensin II-infused animals that had not developed aneurysm. As expected, CD68 (macrophage marker) and several members of MMP family were found to be upregulated in AAA in our model. MMP enzymatic activity in vivo is dependent on MMP [and tissue inhibitors of MMPs (TIMP)] expression as well as MMP activation, a process that exposes the catalytic domain which is the target of RP805. Consistent with its high affinity and specificity for MMP activation, RP805 uptake in vivo closely paralleled aortic MMP activity assessed by zymography ex vivo. The strong correlation between MMP activity and CD68 expression in this study is in accordance with the understanding of the pathobiology of the disease where inflammation and matrix remodeling are two closely related processes. This association was also reflected in the robust correlation between RP805 signal in vivo and CD68 expression. This indicates a potential role of RP805 imaging, not only in depicting MMP activation, but also in assessing inflammation within the vessel wall. Given the current lack of a reliable, specific imaging technique for assessment of inflammation, the potential of MMP imaging in depicting vessel wall inflammation is of interest to investigators, and possibly in the future, clinicians for characterization of inflammatory vascular diseases. To establish a direct link between RP805 uptake and inflammation in aneurysm, future studies should explore the effects of anti-inflammatory interventions on RP805 uptake in AAA.

There is considerable heterogeneity in response to angiotensin II infusion, as some animals do not develop aneurysm, and others develop a large aneurysm or die of rupture. Therefore, we opted not to exclude any animal as outlier. The absence of a correlation between AAA diameter (the conventional clinical risk stratifying criterion) and RP805 uptake excludes the possibility that tissue mass is the major determinant of tracer uptake in AAA. In conjunction with the robust correlation between MMP activity or CD68 expression and MMP tracer uptake in aneurysmal aorta, this lack of correlation with AAA size suggests that RP805 imaging offers the opportunity to measure the underlying pathobiological processes in AAA which are not necessarily reflected in aneurysm size. RP805 microSPECT imaging at one week after initiation of angiotensin II infusion demonstrated that MMP imaging is useful for identifying those aneurysms that will expand or rupture. Indeed, on these early images, the abdominal aorta signal in those mice that ended up developing AAA or die of aneurysm rupture in the following weeks was significantly higher than in those mice that did not develop AAA. Due to limitations of CT imaging, we could not address the potential presence of small AAAs at the time of early imaging. However, we did detect three AAAs on CT images obtained at 1 week, and even after excluding the data from these mice the difference in RP805 uptake between those mice with rupture/AAA and those with no AAA at 4 weeks remained statistically significant (data not shown). In practical terms, any potential future application of MMP-targeted molecular imaging for prediction of expansion or rupture will be limited to those patients with existing AAA.

In conclusion, MMP-targeted molecular imaging can detect MMP activation and vessel wall inflammation in vivo in AAA, and help assess a murine AAA’s propensity for expansion and rupture. Whether the extent and magnitude of inflammation and MMP activation seen in the experimental mouse model correspond exactly with what is seen in human AAA remains to be determined. Ultimately, while the study demonstrates proof of concept in the experimental setting, human studies are necessary to demonstrate the feasibility of MMP-directed imaging for predicting accelerated progression of AAA and also risk of rupture. Clinical translation of MMP imaging can lead to better risk stratification of patients with small AAA to identify the subset that would benefit from for more invasive treatment or more frequent surveillance. It can also help spare those patients with large, but stable AAA from the unnecessary risk associated with invasive aneurysm repair. Finally, beyond its potential role in AAA characterization, MMP-targeted imaging can potentially help the development and monitoring of the effects of emerging medical therapies for AAA and other vascular diseases 38.

Supplementary Material

Supplementary Data
Supplementary Table and Figures

Acknowledgments

Funding Sources

This work was supported by National Institutes of Health R01 HL112992, R01 HL114703, and Department of Veterans Affairs Merit Award I0-BX001750.

Footnotes

Disclosures:

Simon Robinson is employee of Lantheus Medical Imaging. Mehran M. Sadeghi received experimental tracers from Lantheus Medical Imaging.

References

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Supplementary Materials

Supplementary Data
NIHMS645652-supplement-Supplementary_Data.pdf (544.4KB, pdf)
Supplementary Table and Figures
NIHMS645652-supplement-Supplementary_Table_and_Figures.pdf (453.7KB, pdf)

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