Author + information
- Received June 30, 2011
- Revision received August 17, 2011
- Accepted September 5, 2011
- Published online December 6, 2011.
- Ahmed Klink⁎,
- Joeri Heynens†,
- Beatriz Herranz, PhD⁎,‡,
- Mark E. Lobatto, MD⁎,§,
- Teresa Arias, PhD⁎,‡,
- Honorius M.H.F. Sanders, PhD†,
- Gustav J. Strijkers, PhD†,
- Maarten Merkx, PhD†,
- Klaas Nicolay, PhD†,
- Valentin Fuster, MD, PhD‡∥,
- Alain Tedgui, PhD¶,
- Ziad Mallat, MD, PhD#,
- Willem J.M. Mulder, PhD⁎ and
- Zahi A. Fayad, PhD⁎∥,⁎ ()
- ↵⁎Reprint requests and correspondence to
: Dr. Zahi A. Fayad, Translational and Molecular Imaging Institute, Mount Sinai School of Medicine, Atran Berg Laboratory Building, Floor 8, Room AB8-826, 1428 Madison Avenue, New York, New York 10029
Objectives The goal of this study was to use noninvasive conventional and molecular magnetic resonance imaging (MRI) to detect and characterize abdominal aortic aneurysms (AAAs) in vivo.
Background Collagen is an essential constituent of aneurysms. Noninvasive MRI of collagen may represent an opportunity to help detect and better characterize AAAs and initiate intervention.
Methods We used an AAA C57BL/6 mouse model in which a combination of angiotensin II infusion and transforming growth factor–β neutralization results in AAA formation with incidence of aortic rupture. High-resolution, multisequence MRI was performed to characterize the temporal progression of an AAA. To allow molecular MRI of collagen, paramagnetic/fluorescent micellar nanoparticles functionalized with a collagen-binding protein (CNA-35) were intravenously administered. In vivo imaging results were corroborated with immunohistochemistry and confocal fluorescence microscopy.
Results High-resolution, multisequence MRI allowed the visualization of the primary fibrotic response in the aortic wall. As the aneurysm progressed, the formation of a secondary channel or dissection was detected. Further analysis revealed a dramatic increase of the aortic diameter. Injection of CNA-35 micelles resulted in a significantly higher magnetic resonance signal enhancement in the aneurysmal wall compared with nonspecific micelles. Histological studies revealed the presence of collagen in regions of magnetic resonance signal enhancement, and confocal microscopy proved the precise co-localization of CNA-35 micelles with type I collagen. In addition, in a proof-of-concept experiment, we reported the potential of CNA-35 micelles to discriminate between stable AAA lesions and aneurysms that were likely to rapidly progress or rupture.
Conclusions High-resolution, multisequence MRI allowed longitudinal monitoring of AAA progression while the presence of collagen was visualized by nanoparticle-enhanced MRI.
Abdominal aortic aneurysms (AAAs) are permanent dilations of the abdominal aorta that exceed the normal diameter by >50% and which present a life-threatening degenerative disease. They occur in 5% to 9% of the population over the age of 65 years and are the tenth leading cause of death in western countries (1). AAA progression is characterized by a long period of asymptomatic growth of the abdominal aorta. Once it reaches 5.5 cm, there is an increased chance of rupture, with a mortality rate >80%. Surgery and endovascular aortic repair (EVAR) are currently the only interventions for patients diagnosed with AAA. These procedures are costly and associated with high morbidity and mortality rates (2). Generally, surgery or EVAR is recommended when the AAA reaches a size of 55 mm or if the expansion rate is >1 cm/year (2). Although currently the best predictor of aneurysm expansion is the baseline size at diagnosis (3), the prognosis remains complex due to the nonlinearity and unpredictability of expansion rates (4). More specific prognostic predictors would offer the chance to better select intervention strategies for individual patients.
Different animal models have been developed in the past several years to improve the understanding of AAA pathophysiology. Recently, Wang et al. (5) introduced a model in which a combination of angiotensin II (Ang II) and anti-transforming growth factor (TGF)-β administered to C57BL/6 wild-type mice leads to AAA formation and the occurrence of fatal AAA rupture as high as 80%. Currently, this is the only animal model that displays such a relevant rate of AAA rupture, thereby enabling monitoring of the mechanisms at play in this fatal process.
The degradation of the extracellular matrix (ECM) in the medial wall is key to the formation, progression, and rupture of AAAs (6). Particularly, the turnover of collagen, an essential component of the ECM, is known to be responsible for the remodeling that occurs in the adventitia. Studies indicate that perturbations in collagen microarchitecture and networks, probably as a result of collagen degradation and inappropriate collagen deposition, may alter vessel wall response to mechanical load and lead to vessel wall failure (7). Therefore, imaging collagen in AAAs might provide valuable information about the state of aneurysm development and the identification of AAAs prone to severely progress or rupture.
In the current work, we first explored the use of multisequence, high-field magnetic resonance imaging (MRI) to characterize the development of AAAs in the aforementioned model. We then applied recently developed fluorescent/paramagnetic nanoparticles (8), functionalized with a collagen-specific protein (CNA-35) (9–13), to identify the presence of collagen in the aneurysmal wall using molecular MRI. Finally, in a proof-of-concept experiment, we studied the potential of CNA-35 micelles to discriminate between stable AAAs and aneurysms prone to progress and eventually rupture.
All the procedures were approved by the Mount Sinai School of Medicine Institutional Animal Care and Use Committee. C57BL/6J male mice (The Jackson Laboratory, Bar Harbor, Maine) between the ages of 8 and 12 weeks were used. As described in more detail by Wang et al. (5), AAAs were induced in wild-type mice by continuous infusion of Ang II at 1,000 ng/kg/min for a maximum of 28 days, along with systemic neutralization of TGF-β. Briefly, animals were anesthetized with isoflurane (4% induction, 1.5% maintenance), and mini-pumps (Alzet, model 2004 DURECT Corporation, Cupertino, California) containing 200 μl of Ang II were implanted subcutaneously. To achieve systemic neutralization of TGF-β, intraperitoneal injections of an anti–TGF-β antibody (dose: 20 mg/kg) were performed every other day, which results in AAA formation in the suprarenal region of the aorta.
Multisequence in vivo MRI of AAA progression
AAAs were induced in a group of mice as described above (n = 3). MRI scanning was performed using a 9.4-T, small-animal, vertical bore MRI scanner (Bruker BioSpec, Bruker, Germany). Each animal was placed in the center of a whole-body coil (35-mm inner diameter), under continuous isoflurane anesthesia, which was positioned in the scanner. The animals were connected to a respiratory rate monitor, and the flow of anesthetic gas was constantly regulated to maintain a breathing rate of 60 breaths/min. Baseline MRI scanning was followed by scans every other day until sacrifice. Each scan session started with a pilot scan with 3 orthogonal slices, followed by high-resolution, black-blood T1-weighted (T1W), T2-weighted (T2W), and proton density–weighted (PDW) spin echo imaging, of which the exact parameters are reported in the online version of this paper. The suprarenal region of the aorta encompassing 22 mm immediately superior to the right renal artery was imaged. Subsequently, a time-of-flight angiography sequence was used to visualize the blood flow and generate a 3-dimensional reconstruction of the aorta to allow visualization of vessel dilation.
Molecular MRI of collagen
AAAs were induced in wild-type mice (n = 10) according to the aforementioned protocol. The presence of an aneurysm was assessed using magnetic resonance (MR) angiography. CNA-35 micelles or the mutant Y175K CNA-35 micelles (12) (dose: 50 μmol Gd/kg) were injected in the tail vein of the animals (n = 5 for each group). The mice were imaged pre- and 32 hours' post-injection to allow proper clearance of the micelles from the circulating blood, using a T1W spin echo sequence, with the same parameters as used for the multispectral imaging group (Online Appendix). To ensure that AAA remodeling remained minimal between pre- and post-imaging sessions, the injections of anti–TGF-β antibody were terminated before the imaging sessions. Animals that received subcutaneous infusion and intraperitoneal injections of saline were injected with CNA-35 micelles and used as controls (n = 4). In addition, the half-life of CNA-35 micelles was investigated by collecting blood samples at different time points over a period of 24 h. The blood samples were imaged on the IVIS optical system (Calliper Life Sciences, Inc., Hopkinton, Massachusetts), and photon counts were measured to determine the half-life.
Predictive value of (molecular) MRI for the assessment of experimental AAA
AAA were induced in C57BL/6 mice (n = 8) as described here (10 mg/kg). To study whether CNA-35 micelles-enhanced molecular MRI can discriminate between stable AAA lesions and aneurysms that are prone to severely progress or rupture, animals underwent MR imaging after intravenous administration of CNA-35 micelles at days 5 and 15 after the onset of AAA. These animals were monitored for death or survival, whereas aneurysm severity (stage I to stage IV) was assessed by looking at aneurysm morphology as previously described (14). Mice that suffered from ruptured aneurysms were classified as stage IV.
Image analysis and statistics
Pre- and post-MR images were analyzed using OsiriX DICOM Viewer (Geneva, Switzerland). Each slice was co-registered using the spinal cord as a landmark. AAAs were identified on the pre-injection images. A region of interest was drawn around the aneurysmal wall, and the average signal intensity was measured (Iwall) over 10 slices throughout the aneurysm. In addition, regions of interest were also drawn in the surrounding muscle as well as in the noise, and the average signal intensity and SD of the noise were measured, respectively (Imuscle, σnoise). Signal-to-noise ratios were calculated for the wall and the muscle (, ). The contrast-to-noise ratio was calculated () and further normalized to the pre-injection values (). Finally, the normalized percentage signal enhancement (%NSE) was determined as %NSE = (CNRNorm − 1) × 100. The data are presented as mean ± SD.
One-way and 2-way analysis of variance (ANOVA) tests were performed to determine statistical significance. A post-hoc Tukey test was performed to compare each of the 3 groups injected with CNA-35 micelles or mutant CNA-35 micelles. A Bonferroni post-hoc test was performed to compare the %NSE of stages II, III, and IV aneurysms at days 5 and 15 of AAA development. A p value <0.05 was considered statistically significant. Statistical analysis was performed using PRISM (GraphPad Software, La Jolla, California).
Immunohistochemistry and confocal microscopy
Animals were sacrificed by an isoflurane overdose. Transcardiac perfusions through the left ventricle were conducted with saline and paraformaldehyde (4%). The suprarenal aorta was excised, collected in optimum cutting temperature medium (OCT), and frozen for further histological examination. Combined Masson elastin (CME) staining was performed to stain for collagen and elastin fibers. In addition, type I collagen and macrophage CD68 staining was performed using a primary rabbit anti-mouse type I collagen antibody and a primary rat anti-mouse CD68 antibody, respectively (Santa Cruz Biotechnology, Inc., Santa Cruz, California); the primary antibodies were detected using appropriate secondary antibodies coupled to an Alexa 647 fluorophore. For each animal, 10 representative sections were chosen, fixed in 4% paraformaldehyde, and washed twice in phosphate-buffered saline. The sections were blocked with 2% goat serum in phosphate-buffered saline for 20 min at room temperature. After blocking, the sections were incubated with the type I collagen primary antibody or CD68 primary antibody (1:100 dilution) for an hour. After washing with phosphate-buffered saline, the secondary antibody was added for 30 min. Finally, the autofluorescence of the tissue was reduced using copper sulfate, and sections were mounted with DAPI-containing VECTASHIELD mounting medium (Vector Laboratories Inc, Burlingame, California) and sealed with coverslips, shielded from light, and kept at 4°C until confocal laser scanning microscopy (CLSM) was performed within 48 h. Sections incubated with the secondary antibody only were used as negative controls. CLSM imaging was performed with a Leica Sp5DM microscope (Leica Microsystems, Wetzlar, Germany). Adjacent sections were stained for type I collagen and macrophages to study their respective localization with regard to the rhodamine-labeled CNA-35 micelles.
Characterization and in vitro binding experiments
The synthesis and a schematic of the paramagnetic micelles are included in the Online Appendix (Online Fig. 1A). The size of the micelles before conjugation was 14 ± 1.5 nm, whereas the CNA-35 and mutant CNA-35–coupled micelles had a mean size of 25 ± 3 nm. Phosphate determination and protein determination yielded a lipid:protein ratio of approximately 50:1, corresponding to approximately 2 proteins per micelle. The ionic relaxivity, a measure of MRI contrast–generating potency (15), of bare, CNA-35, or mutant CNA-35–conjugated micelles was 12 mM–1s–1.
CNA-35 is a natural collagen-binding protein that has been shown to have affinity for collagen fibers (11). Indeed, collagen fibers contain a high density of binding sites with both high and low affinity for the CNA-35 protein, resulting in an average constant dissociation value of 500 nm (11). Our in vitro binding experiment (described in the Online paper) corroborated these previously published findings (11). We observed an apparent binding of the CNA-35 micelles to collagen after extensive washing, whereas little binding occurred for mutant CNA-35 or unconjugated micelles (Online Fig. 1B). A second in vitro experiment was performed to further confirm these findings and to investigate the collagen binding of Cy5.5-labeled CNA-35 micelles with near-infrared fluorescence (NIRF) imaging using the IVIS optical imaging system. Similar to the plate reader studies, we found a clear binding of the CNA-35 micelles to collagen (Online Fig. 1C). Finally, the blood circulation half-life of the CNA-35 micelles in male C57BL/6 mice (n = 2) was determined to be 4.3 h.
Multisequence in vivo imaging of AAA progression
AAAs were induced in male C57BL/6 mice through continuous infusion of Ang II and administration of anti–TGF-β every other day. The abdominal region of these mice was imaged using high-resolution, multisequence MRI to monitor the temporal progression of the AAA at the anatomical level. T1W, T2W, and PDW MRI, as well as MRI angiography, were performed. Multisequence MRI allowed the visualization of the primary fibrotic response in the aortic wall at day 3. It also revealed luminal stenosis and medial rupture, with blood infiltrating the adventitial tissue 6 days after AAA induction (Figs. 1A to 1C). At day 8, the adventitia appeared to be extensively destructed, with the formation of a double channel clearly visible on different sequences. The 3-dimensional reconstruction resulting from the time-of-flight angiography confirmed the aortic dilation and the presence of blood infiltrating the adventitial tissue (Fig. 1D, arrow). Additional analysis revealed a dramatic increase in the diameter of the aorta, reaching 50% growth at day 5 and up to a 150% growth on day 15 (Fig. 1F). To corroborate the in vivo findings, immunohistological examination of the aortas was performed at the animals' death. The results confirmed the presence of AAAs and allowed the precise validation of several features of vessel wall remodeling observed by using in vivo MRI. An extreme degradation of the ECM was observed due to massive destruction of adventitial collagen. Only a thin cap remained visible on the outer edge of the aneurysmal wall, preventing the vessel from rupturing. Intense degradation of the elastin fibers as well as the complete rupture of the medial layer of the aorta was clearly identified (Fig. 1E, arrow). Due to the rupture, blood originating from the aortic lumen was able to infiltrate the adventitia, thereby forming a secondary lumen referred to as false channel or dissection (Fig. 1E). The resulting increase of the aortic diameter was visibly noted.
In vivo molecular imaging of collagen
Although high-resolution, multisequence MRI allows the characterization of AAA progression on an anatomical level, it does not provide any information on the underlying molecular biological processes. Therefore, we decided to evaluate the presence of collagen in the same mouse model because it represents a key constituent of aneurysms. To that end, collagen-specific CNA-35 micelles were synthesized and injected for nanoparticle-enhanced MR imaging. Before the injection of micelles, the presence of aneurysms was evaluated using MR angiography. T1W MR images were then acquired before and 32 hours after injection of CNA-35 or mutant CNA-35 micelles. In mice injected with CNA-35 micelles, a clear signal enhancement in the aneurysmal region was observed (Fig. 2A), whereas a significantly lower signal enhancement was visible after the injection of mutant CNA-35 micelles. As shown in Figure 2B, data analysis of the MR images revealed an 80.15 ± 37% increase in %NSE after injection of CNA-35 micelles compared with a 30.90% ± 19% increase after the injection of mutant CNA-35 micelles (n = 5; p = 0.0048, 1-way ANOVA test; p < 0.05, post-hoc Tukey test). No enhancement was observed in healthy animals injected with CNA-35 micelles.
Examination of the corresponding histological slices stained with CME revealed an advanced degree of adventitial destruction with the presence of collagen in the areas of MR signal enhancement (Figs. 3B,3C, 3F, and 3G). Importantly, further examination by confocal microscopy demonstrated the co-localization of the CNA-35 micelles (red) with collagen-I (green) in the adventitia (Fig. 3D). In animals injected with mutant CNA-35 micelles, the fluorescent signal originating from the micelles was found to be nearly nonexistent in the regions that stained positively for type I collagen (Fig. 3H). In addition, CLSM images of macrophage stained with a CD68 antibody revealed no association between macrophages and CNA-35 or mutant CNA-35 micelles (data not shown). It is noteworthy that mutant CNA-35 micelles were not associated with any specific biological structures. In addition, the collagen MR signal enhancement pattern observed in different animals allowed differentiation between collagen-rich and collagen-poor lesions as observed on corresponding CME stains (Fig. 4).
Ex vivo fluorescence imaging
To corroborate the CNA-35 micelles-enhanced in vivo MRI results and to investigate their spatial distribution in the aorta, Cy5.5-labeled micelles were injected in mice after AAA development as well as in healthy animals. NIRF imaging, a so-called hotspot imaging technique (16), allows the visualization of the Cy5.5-labeled micelles with high sensitivity in intact aortas (17,18). Mice were sacrificed 32 hours post-injection, and the excised aortas were imaged with NIRF imaging. Cy5.5-labeled CNA-35 micelles were clearly observed in the AAA region (Online Fig. 2). In comparison, healthy animals and noninjected AAA animals marginally displayed fluorescent signals, further supporting the specific binding of CNA-35 micelles in the aneurysmal region.
Predictive value of (molecular) MRI for the assessment of experimental AAA
As described here, the injection of CNA-35 micelles allowed the discrimination between collagen-poor and collagen-rich lesions (Fig. 4). It has been shown in the literature that low levels of collagen are associated with AAA progression and ultimately rupture (7). Hence, we hypothesized that CNA-35 micelles have the potential to discriminate between stable AAA lesions and aneurysms that are prone to progress or rupture. We imaged CNA-35–injected animals at days 5 and 15 of AAA development and subsequently monitored them for survival. On death or sacrifice, the aneurysm severity was evaluated by looking at the aneurysm morphology as previously described (14). Administration of anti–TGF-β in Ang II–infused mice led to mortality from aneurysm rupture (stage IV) in 3 mice, whereas the remaining 5 animals survived bearing stage II and III AAAs. All the main features of human aneurysms ranging from the fibrotic response around the vessel wall to signs of medial dissection and formation of a secondary channel were observed.
As shown in Figure 5, animals that were morphologically classified with stage II aneurysms displayed marked enhancement after CNA-35 administration at days 5 and 15 of aneurysmal development, suggesting a collagen-rich response of the aortic wall. These lesions were stable, as no aortic dissection and medial rupture were observed, and the animal survived the 28-day period of Ang II + anti–TGF-β administration. In contrast, all the mice retrospectively classified with stage III and IV aneurysms (n = 5) exhibited a dissection of the media at day 5 (Fig. 5A, arrow) and showed a marked decrease of approximately 30% in %NSE in both groups relative to stage II AAAs (48.50 ± 4.950 vs. 72.01 ± 10.47 [p > 0.05] and 46.68 ± 7.652 vs. 72.01 ± 10.47 [p > 0.05], respectively) (Fig. 5B). Imaging at day 15 showed a critical progression of AAA development with an extensive remodeling of the vessel wall and presence of a secondary channel. Ruptured stage IV aneurysms showed little signal enhancement after CNA-35 injection, suggesting a lower collagen content as opposed to nonruptured stage II and III aneurysms (52.13 ± 8.408% vs. 120.7 ± 19.49% [p < 0.001] and 52.13 ± 8.408% vs. 87.51 ± 3.521% [p < 0.05], respectively) (Fig. 5B). Consistent with this hypothesis, histological examination on the death of the animals at days 19, 22, and 23 revealed substantial collagen degradation in fatal aneurysms compared with viable stage II and III AAAs. A 2-way ANOVA test revealed that both factors (stage of aneurysm and time) significantly alter %NSE (p = 0.0027 and p = 0.0024, respectively) and that there is a significant interaction between the 2 factors (p = 0.0407). These findings show a potential for CNA-35 micelles to discriminate between stable AAA lesions with important collagen content and aneurysms that are likely to rapidly progress and eventually rupture.
In the current study, we demonstrated the ability of high-resolution conventional and molecular MRI to detect key anatomical features of aneurysmal progression and to identify the presence of collagen in the aneurysmal wall in a new AAA mouse model. Conventional multisequence black-blood MRI permitted the visualization of the different stages of AAA progression. Over the course of aneurysm development, we reported the accurate identification of the primary fibrotic response in the vessel wall with consequent luminal stenosis. As the aneurysm progressed, we visualized the medial rupture with blood infiltrating the remodeled adventitia, which resulted in the formation of a double channel. The ensuing increase in aortic diameter was clearly noted and quantified. Histological examination on the death of the animals matched the MR observations and confirmed the presence of the aforementioned landmarks of aneurysmal progression.
Molecular MRI, via intravenous administration of collagen-targeted CNA-35 micelles, provided valuable information about the presence of collagen in the aneurysmal wall. The injection of CNA-35 micelles resulted in a significantly higher MR signal enhancement than the injection of mutant CNA-35 micelles. Histological staining for collagen in corresponding sections confirmed the presence of collagen in the areas of MR signal enhancement. Additional examination with fluorescent confocal microscopy demonstrated the precise co-localization of CNA-35 micelles with collagen stained in the remodeled adventitia. Importantly, in a proof-of-concept experiment, CNA-35 micelle-enhanced MRI allowed the differentiation between stable AAA lesions and aneurysms that are likely to rapidly progress and rupture.
Imaging technologies in general, and ultrasound imaging in particular, are currently used to obtain information related to AAA in humans (19,20). An angiography is routinely performed to obtain preoperative anatomical information on the aneurysm and its relationship to surrounding structures (21). However, disadvantages include the use of contrast and exposure to radiation. As for ultrasound imaging, it is widely used to monitor AAA progression, but it provides little contrast and suffers from a poor signal-to-noise ratio, which limits the ability to obtain a detailed evaluation of the remodeled adventitia, particularly in preclinical studies involving small-animal models. Although some recent reports have shown the ability of ultrasound Doppler to identify medial rupture and differentiate between the lumen and the false channel in mice (5), the resolution of ultrasound remains subpar compared with MRI.
Recently, MRI has been used successfully by Turner et al. (22) to assess the development of an Ang II–induced aneurysm model in hypercholesterolemic mice. The investigators revealed that MR measurements of the AAA area performed on a bright-blood sequence were precisely correlated with the AAA area measured by using histology. In the current study, the aortic diameter measured with 3 different MR methods (T1W, T2W, and PDW) was evaluated at each imaging time point. The results showed a fast increase of the aortic diameter reaching 50% (the minimum value to define an aneurysm) 5 days after the initiation of the experiment on average. In addition to providing aortic diameter measurements, multisequence black-blood imaging offered superior contrast compared with bright-blood sequences for the examination of vessel wall remodeling during AAA formation and progression. Furthermore, it allowed the accurate identification of the medial rupture and formation of a double channel, similar to bright-blood imaging. Particularly on the T1W sequence, the medial rupture was clearly identifiable as soon as 3 days after AAA induction compared with the T2W and PDW sequences, which did not allow such evident visualization until 6 days after aneurysm initiation (Fig. 1B). In addition, multisequence black-blood imaging also offered adequate resolution to image the medial-adventitial interface. Indeed, despite various degrees of aortic stenosis caused by different degrees of collagen degradation and thrombus formation, the lumen always remained easily distinguishable from the remodeled adventitia, even on blood extravasation in the adventitial space. Multisequence black-blood MR imaging offers the benefit of increased accuracy in characterizing the anatomical features of AAA temporal progression.
One of the biological processes underlying AAA formation and progression is the degradation of the ECM, particularly collagen. Members of the matrix metalloproteinase and cysteine protease families can degrade the highly protease-resistant structures of type I and type III collagen (23–25). After the destabilization of the collagen structure, other less specific proteases can further degrade collagen. This increased collagen degradation in combination with inadequate collagen deposition has been associated with the progression and ultimately rupture of AAAs. Therefore, imaging collagen might offer additional insights about the state of aneurysm progression. CNA-35 consists of 2 N domains (N1 and N2) from a collagen adhesion protein originating from Staphylococcus aureus and was used as a targeting ligand. Its specificity for collagen was previously demonstrated in vitro and in vivo (9,10,12,13). In its open conformation, the CNA-35 protein can fold and lock around the collagen fiber (11). This binding process implies that CNA-35 micelles would also adhere to individual triple helical fibers of collagen, which are hardly present in healthy tissue, in addition to binding to mature collagen fibers. Degraded collagen and newly formed collagen fibers, highly present in advanced stages of AAA, are associated with high portions of free triple helices. Therefore, the bright signal enhancement observed in the aneurysmal wall of animals injected with CNA-35 micelles might derive from a combination of the micelles' increased affinity to mature collagen fibers as well as to unstructured collagen present in AAAs (7). When CNA-35 micelles were injected in animals in healthy animals, no MR signal enhancement was observed (Fig. 2B). Given the size of these particles (25 nm), their inability to penetrate healthy tissue is likely to be the limiting factor to collagen binding.
It is apparent that the degree of adventitial collagen degradation relates to the stage of aneurysmal severity. In fact, it has been shown previously that increased collagen degradation is associated with AAA progression and ultimately rupture (7). Thus, one could argue that increased level of collagen in the aortic wall stabilizes the aneurysm. We therefore hypothesized that CNA-35 micelles would allow the distinction between stable collagen-rich AAA lesions and collagen-poor lesions that are more likely to progress and rupture. To that end, we designed a proof-of-concept experiment in which the normalized signal enhancement relative to CNA-35 injection was calculated at early and late stage of AAA development and correlated with the aneurysm severity determined by their morphology (stage I to IV). Our findings revealed the ability of CNA-35 micelles to identify stable aneurysms, as the collagen-rich response of the aortic wall of noncomplicated lesions was recognizable with a high NSE percentage value (Fig. 5). In addition, a decrease of CNA-35 uptake at early stage of aneurysmal development (day 5) in stage III and IV aneurysms was associated with a rupture of the medial layer of the aorta and subsequent critical AAA progression. Interestingly, the medial rupture seemed to occur in the areas of lowest CNA-35 uptake (Fig. 5, arrow). Importantly, lesions with the lowest %NSE value that did not increase at follow-up were identified as fatal stage IV aneurysms and presented critical collagen degradation compared with viable stage III aneurysms.
Although collagen is likely not the only biological component at play in AAA progression and rupture, its precise monitoring may benefit diagnosis and staging of the aneurysm. Our results show that CNA-35 micelle-enhanced MRI represents an extremely interesting concept for the detection of collagen in the aneurysmal wall and has potential for predicting the course of AAA development. However, because of the low number of animals per group in that experiment, more extensive studies are necessary to confirm the predictive value of CNA-35 micelle-enhanced MRI in assessing AAA progression/rupture.
The current clinical guidelines (26) for the management of aneurysms are mainly based on the size of aneurysms and recommend repeated anatomical imaging every 3 to 6 months for aneurysms >4 cm. Endovascular repair or surgery is recommended for AAAs that are >5.5 cm, symptomatic, or fast growing. Intervention is not indicated when these criteria are not met as the chance of rupture is <2.1% (25). The high rate of mortality associated with reparative surgery would expose patients to unnecessary risks. Noninvasive imaging approaches, such as high-resolution multisequence black-blood MRI and collagen-targeted MRI described in this study, could ultimately improve the identification of patients at risk and facilitate the clinical management of AAAs (27). It is noteworthy that other biological targets involved in AAA pathology are also persuaded as imaging targets in an effort to improve the characterization of the aneurysmal wall. Nahrendorf et al. (28) developed a macrophage-targeted nanoparticle for positron emission tomography–computed tomography detection of inflammation in aortic aneurysms. Recently, a pilot study used ultrasmall superparamagnetic iron oxide nanoparticles uptaken to predict aneurysm growth in humans (29). In addition, such anatomical and biological information obtained may replace the preoperative need of a computed tomography angiography.
We revealed that high-resolution, multisequence MRI allowed the visualization of critical features of vessel remodeling inherent to AAA formation and progression, whereas collagen-targeted nanoparticle-enhanced MRI permitted the noninvasive detection of collagen, an essential component in aneurysm pathology.
The authors acknowledge Dr. Rolando Nolasco for his work on the histological samples.
For supplementary materials (including tables and figures), please see the online version of this article.
This study was funded by the Netherlands Heart Foundation project number 2009SB006, the collaborative project ATHIM (Atherothrombosis Molecular Imaging), NIH/NHLBIR01HL070121, and NIH/NIBIBR01EB009638 (Dr. Fayad). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- abdominal aortic aneurysm
- angiotensin II
- confocal laser scanning microscopy
- combined Masson elastin
- extracellular matrix
- endovascular aortic repair
- magnetic resonance
- magnetic resonance imaging
- near-infrared fluorescence
- normalized percentage signal enhancement
- proton density–weighted
- tumor growth factor
- Received June 30, 2011.
- Revision received August 17, 2011.
- Accepted September 5, 2011.
- American College of Cardiology Foundation
- Michel J.B.
- Powell J.T.,
- Brady A.R.
- Zong Y.,
- Xu Y.,
- Liang X.,
- et al.
- Manning M.W.,
- Cassi L.A.,
- Huang J.,
- et al.
- Sosnovik D.E.,
- Nahrendorf M.,
- Deliolanis N.,
- et al.
- Turner G.H.,
- Olzinski A.R.,
- Bernard R.E.,
- et al.
- Lutgens S.P.,
- Cleutjens K.B.,
- Daemen M.J.,
- Heeneman S.
- Hirsch A.T.,
- Haskal Z.J.,
- Hertzer N.R.,
- et al.
- Richards J.M.,
- Semple S.I.,
- Macgillivray T.J.,
- et al.