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Published online before print November 21, 2007, 10.1148/radiol.2461070032
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(Radiology 2008;246:185-192.)
© RSNA, 2007

Molecular Imaging

Aortic Aneurysms in a Rat Model: In Vivo MR Imaging of Endovascular Cell Therapy1

Jean-François Deux, MD, PhD, Jiangping Dai, MD, PhD, Charlotte Rivière, PhD, Florence Gazeau, PhD, Philippe Méric, PhD, Brigitte Gillet, PhD, Jacky Roger, MD, PhD, Jean-Noel Pons, PhD, Didier Letourneur, PhD, Frank P. Boudghène, MD, PhD, and Eric Allaire, MD, PhD

1 From INSERM U 698, Bioengineering Department, University Paris 7-University Paris 13, X. Bichat Hospital, Paris, France (J.F.D., D.L.); Radiology Department (J.F.D.) and Centre National de la Recherche Scientifique, Unité Nixte de Recherche 7054 (J.D., E.A.), Assistance Publique-Hôpitaux de Paris, IFR de Médecine, University Paris 12, H. Mondor Hospital, Créteil, France; Laboratoire Matière et Systèmes Complexes, Paris, France (C.R., F.G.); Laboratoire de Resonance Magnetique Nucleaire Biologique-Institut de Chimie des Substances Naturelles, Gif sur Yvette, France (P.M., B.G.); Laboratoire des Liquides Ioniques et Interfaces Chargées, Paris, France (C.R., J.R., J.N.P.); and Radiology Department, Tenon Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France (F.P.B.). Received January 7, 2007; revision requested March 2; revision received April 16; accepted May 7; final version accepted June 18. Supported by the Centre National de la Recherche Scientifique (CNRS), the French Institut National de la Santé et de la Recherche Médicale (INSERM), the Ministère de l'Education Nationale de l'Enseignement Supérieur et de la Recherche (Action Concertée Incitative technologies pour la santé), Université Paris 13 (Bonus qualité recherche and Institut Federal de Recherche Paris-Nord Plaine de France), University Paris 12 Val de Marne, the Fondation Bettencourt-Schueller (Prix Coup d'Elan), and the Fondation de la Recherche Médicale. C.R. supported by the French Direction Générale de l'Armement (DGA doctorat fellowship). Address correspondence to J.F.D. (e-mail: jean-francois.deux{at}hmn.aphp.fr).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
 References
 
Purpose:To prospectively evaluate in rats whether magnetic cell labeling can be used to noninvasively assess the technical success of endovascular cell therapy for abdominal aortic aneurysms (AAAs).

Materials and Methods:The study was approved by an institutional animal care and use committee. Vascular smooth muscle cells (VSMCs) labeled with iron oxide nanoparticles were seeded endovascularly in already formed AAAs. T2*-weighted gradient-echo and T2-weighted spin-echo magnetic resonance (MR) imaging was performed in vivo at 1.5 T before and 30 minutes after the injection of iron-loaded VSMCs (14 rats), nonlabeled VSMCs (three rats), or iron-free particles (three rats). Ten rats were euthanized shortly after the injection (day 0). Of the 10 remaining rats, which were seeded with iron-loaded cells, three were imaged on day 7 after cell delivery; three, on day 14; and four, on day 28; then they were euthanized. Ex vivo high-field-strength MR imaging of two AAAs was performed 28 days after cell delivery. Histologic examination of cross sections of all AAAs was performed. Statistical evaluations were performed with a nonparametric Wilcoxon correlation test.

Results:Magnetic cell labeling did not alter the capability of VSMCs to stabilize the diameter of the aneurysms. T2*-weighted gradient-echo images showed areas of hypointense signal within the aortic wall immediately and up to 1 month after cell therapy. The mean signal intensity decreased significantly after cell delivery (from 2362 ± 244 [standard deviation] before to 434 ± 275 after delivery, P < .001). Areas of hypointense signal and iron-loaded VSMCs were colocalized in the area of aortic wall reconstruction at both high-field-strength MR imaging and histologic analysis.

Conclusion:MR imaging with magnetic cell labeling can be used to document endovascular cell delivery in already formed AAAs in rats.

© RSNA, 2007


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
 References
 
Abdominal aortic aneurysms (AAAs) carry a risk of rupture and death that is proportional to the external diameter of the aneurysm (1). Current treatments for AAAs involve the replacement or strengthening of the dilated aorta with prostheses (2) or endoprostheses (3), respectively.

AAAs form as a result of wall thinning, with rarefaction of vascular smooth muscle cells (VSMCs) occurring owing to apoptosis. Disruption of the fibrillar extracellular matrix is driven by an excess of proteases (1,4). In a rat model of already-formed AAAs, endovascular seeding of syngenic VSMCs corrects the wall atrophy by stopping the wall destruction and triggering the development of new aortic tissue rich in {alpha}-actin–positive cells surrounded by an extracellular matrix (5). As a result, the external diameter of an experimental AAA is stabilized.

Magnetic resonance (MR) imaging has been validated for assessment of the diameter (6), wall thickness (7), and mechanical properties (8) of the aorta. MR signal is altered by superparamagnetic compounds, which can be internalized by different cells (9). It has been reported that VSMCs, after being injected into rat myocardium, can be magnetically labeled in vitro by using anionic nanoparticles and can be imaged ex vivo with MR imaging (10). Thus, the purpose of our study was to prospectively evaluate in rats whether magnetic cell labeling can be used to noninvasively assess the technical success of endovascular cell therapy for AAAs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
 References
 
Synthesis and Characterization of Anionic Magnetic Nanoparticles
Our study was approved by the institutional animal care and use committee of H. Mondor Hospital. Stable colloidal suspensions of negatively charged maghemite nanoparticles were synthesized, as described elsewhere (11). At 1.5 T and 25°C, the longitudinal and transverse relaxivities of anionic nanoparticles dispersed in water are 10.2 and 357 L·mmol–1·sec–1, respectively (12).

VSMC Culture and Labeling
VSMCs were isolated from five male Fisher rats (Iffa Credo, Paris, France) (13). The VSMCs were labeled by means of nanoparticle internalization, as described elsewhere (12). Iron oxide nanoparticle–labeled (IONP) VSMCs were resuspended in culture medium. Fourteen aliquots of 106 IONP VSMCs were prepared. Red fluorescent dye (PKH26; Sigma, St Louis, Mo) was used to fluorescently label four of these aliquots. Three aliquots of 106 non–magnetically labeled (control) VSMCs were also prepared; one of them was fluorescently labeled. The iron content in the VSMCs was quantified by using magnetophoresis (11).

Semiquantification of Messenger RNA with Reverse Transcription Polymerase Chain Reaction Assay
Because AAA stabilization after endovascular VSMC seeding is attributed in part to the down regulation of metalloproteinase activity (14), the messenger RNA levels of metalloproteinase in the IONP VSMCs were assessed by means of reverse transcription polymerase chain reaction assay, as described elsewhere (5).

Animals and Surgery
Twenty abdominal aortas decellularized with sodium dodecylsulfate (Sigma) were retrieved from 20 Hartley guinea pigs (Iffa Credo). Each of the 20 tubes of extracellular matrix was implanted orthotopically in a receiver male Fisher rat, so a total of 20 rats received an implant. As described previously (15), the implanted xenograft degenerates into an aneurysm as a consequence of xenogenic extracellular matrix rejection (Fig 1). A second surgical procedure was performed 14 days later (Fig 1), when the xenograft diameter had increased more than 50%. Blood flow in the aneurysm was stopped by means of clamping. A PE10 catheter (Transonic Systems, Maastricht, the Netherlands) was introduced by means of aortotomy performed downstream in the native aorta (5).


Figure 1
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  		Figure 1: Experimental in vivo MR imaging scheme.

 
One investigator (J.D.) injected the IONP and red fluorescent dye–labeled VSMCs—106 cells per AAA—through the PE10 catheter into the lumen of 14 experimental AAAs (in 14 rats). Three additional rats received nonlabeled VSMCs (106 cells per AAA), and three other rats received free anionic iron oxide nanoparticles in suspension (1.0 mmol/L iron). The VSMCs were allowed to attach to the aortic wall by means of sedimentation during clamping. In all but one rat, the VSMCs were injected during four 90° animal rotations (2 minutes per position) to achieve circumferential seeding of cells. The one rat was not rotated during cell delivery so that hemicircumferential delivery of cells could be achieved, as previously reported (16). Blood flow was reestablished after 8 minutes.

In Vivo MR Imaging Protocol
In vivo MR imaging was performed with a clinical 1.5-T MR unit (Signa 1.5 T; GE Medical Systems, Milwaukee, Wis) by using a homemade cylindric coil with a diameter of 120 mm. Transverse T2*-weighted gradient-echo (16/2.8 [repetition time msec/echo time msec], 80-mm field of view, 30° flip angle, 256 x 256 matrix, 2-mm section thickness) and T2-weighted spin-echo (2000/56, 60-mm field of view, 256 x 256 matrix, 2-mm section thickness) sequences were performed.

MR imaging was performed in all 20 rats immediately before and less than 30 minutes (day 0) after cell seeding. Ten rats—four (three rotated and one not rotated during cell delivery) with magnetically labeled cells, three with non–magnetically labeled cells, and three with free iron oxide nanoparticles—were euthanized immediately after cell seeding. The remaining 10 rats, all of which received magnetically labeled VSMCs, were followed up with MR imaging beyond day 0 after cell delivery: A third MR examination was performed in three rats on day 7, in three rats on day 14, and in four rats on day 28. One of these 10 rats was imaged on days 0, 21, and 28 after cell delivery. All rats were euthanized after the final MR examination.

Ex Vivo High-Field-Strength MR Image Acquisition
Ex vivo high-spatial-resolution MR imaging of two AAAs, excised from two rats, was performed with a 9.4-T magnet (Varian Inova; Varian, Palo Alto, Calif) 28 days after endovascular delivery of the magnetically labeled VSMCs. Transverse T2*-weighted gradient-echo imaging of these aneurysms was performed by using 100/3, a 30-mm field of view, a 25° flip angle, a 256 x 256 matrix, and an 80-µm section thickness.

In Vivo MR Imaging Evaluation
For each animal, the maximal aneurysm diameter was measured in vivo at T2-weighted spin-echo MR imaging. On the corresponding histologic section, the boundaries of the aortic wall were defined in consensus between two authors (J.F.D., C.R., 6 and 4 years experience in vascular MR imaging, respectively). Signal intensity was measured by contouring the aortic wall on T2*-weighted gradient-echo images on days 0, 7, 14, and 28 after cell delivery by using the software available on the MR unit. The lumen was excluded from the region of interest. The percentage change in signal intensity was normalized to the signal intensity of the aortic wall before cell delivery. Visual analysis included assessment of heterogeneity and homogeneity, circumferential or focal areas of low signal intensity, and number of distinct areas of hypointense signal detected with MR imaging. The same two authors compared the location of the low-signal-intensity areas with the location of the main foci of Perls Prussian blue stain–positive cells in consensus. Repartition was judged to be concordant if the low-signal-intensity areas and iron-loaded cells seemed to be colocalized.

Histologic and Immunochemical Analyses
The harvested aortas were gently rinsed, fixed in 70° ethanol, and embedded in paraffin. Five-micrometer cross sections were obtained at the level of maximal dilatation. One investigator (J.F.D.) measured the maximal diameter by using a grid in the microscope eyepiece. Sections were stained with eosin and Perls Prussian blue dye for iron detection. A senior laboratory technician with 12 years experience in histologic and immunochemical analyses performed immunochemical analysis by using primary monoclonal mouse antirat antibodies: ED1 (Dako, Copenhagen, Denmark) for monocytes and macrophages and anti–{alpha}-actin (Neomarker, Fremont, Calif) for VSMCs.

Statistical Analysis
Quantitative results for the animal groups were expressed as means and value ranges for diameter and as means ± standard deviations for signal intensity. Comparison of the MR signal intensity measurements before with those after cell or free iron particle seeding within the aortic wall of the 20 rats was performed by using the nonparametric Wilcoxon test (StatView; Abacus Concepts, Berkeley, Calif). P < .005 was considered to indicate significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
 References
 
Magnetic Labeling of VSMCs in Culture
VSMCs incubated for 2 hours with anionic iron oxide nanoparticles (ie, IONP VSMCs) displayed no cell toxicity (>95% exclusion at trypan blue testing). The mean intracellular iron load was 3.9 pg per cell ± 0.9. There were no significant differences at any time point in messenger RNA levels of metalloproteinase 2, metalloproteinase 9, tissue inhibitor of metalloproteinase 1, or tissue inhibitor of metalloproteinase 3 between the IONP VSMCs and the nonlabeled (control) VSMCs (data not shown).

In Vivo MR Imaging on Day 0
Before the injection of the magnetically labeled VSMCs, the aortic wall appeared as a ring of intermediate homogeneous signal intensity with high-signal-intensity blood flow on the T2*-weighted gradient-echo images (Fig 2, A1–A4). Immediately after delivery of the IONP VSMCs (day 0), the T2*-weighted gradient-echo images showed circumferential areas of hypointense signal within the wall of the AAA in all rats that were rotated 90° during cell delivery (Fig 2, B1). In three of the 20 animals, areas of hypointense signal were also detected next to the aortic wall and thus were suggestive of leaks around the aorta during cell delivery (Fig 2, B1). Fluorescent VSMCs were visualized along the walls of the harvested aortas at fluorescent microscopy, confirming efficient cell delivery (Fig 2, inset in B2 and B3). In the one AAA that was seeded with IONP VSMCs without animal rotation, the area of hypointense signal was localized only at the posterior declivis of the aorta (Fig 2, B2). Neither local delivery of non–magnetically labeled VSMCs (Fig 2, B3) nor injection of iron oxide nanoparticles without cells (Fig 2, B4) induced signal intensity changes on the T2*-weighted gradient-echo images.


Figure 2
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  		Figure 2: Transverse in vivo 1.5-T T2*-weighted gradient-echo MR images (16/2.8, 30° flip angle) of AAAs before (A1–A4) and after delivery of IONP VSMCs (B1), fluorescence- and iron-labeled VSMCs (B2), fluorescence-labeled VSMCs (B3), and free iron particles (B4). Note intermediate signal intensity of aneurysm wall before VSMC injection (A1–A4). Vessel lumen appears as bright area (*). B1 shows areas of hypointense signal (black arrows) next to aneurysm wall after delivery of IONP VSMCs. Signal intensity of aortic lumen appears darker probably because of susceptibility artifacts induced by seeded magnetically labeled cells. Low-signal-intensity areas (white arrow) were depicted above aortic wall in three rats, suggesting leaks around aorta during cell delivery. B2, Area of hypointense signal (arrow) was detected in only half the aortic wall when rat was not rotated during cell seeding. No signal intensity change was detected after delivery of non–iron-labeled VSMCs (B3) and free iron nanoparticles (B4). Fluorescence labeling revealed fluorescent cells next to aortic wall after delivery of VSMCs labeled with both iron and red fluorescent dye (B2 inset) or red fluorescent dye only (non–iron labeled, B3 inset). Scale bar = 1 mm.

 
Quantitatively, the mean signal intensity (expressed in arbitrary units) on day 0 after IONP VSMC delivery was significantly decreased on the T2*-weighted gradient-echo images: from 2362 ± 244 to 434 ± 275 (P < .001). The mean signal intensity on the T2-weighted spin-echo images was not significantly modified after cell seeding: 2245 ± 176 before versus 2128 ± 120 after cell delivery.

In Vivo Follow-up
The circumferential areas of hypointense signal detected at MR imaging on day 0 after IONP VSMC injection were identified in all AAAs on the T2*-weighted gradient-echo images from day 7 to day 28 (Fig 3). No signal intensity modification was detected on the T2-weighted spin-echo images (data not shown). The mean signal intensity seen in the aortic wall on the T2*-weighted gradient-echo images increased from day 0 (403 ± 198) to day 7 (912 ± 311) in three rats, from day 0 (240 ± 50) to day 14 (1313 ± 271) in three rats, and from day 0 (323 ± 84) to day 28 (1426 ± 192) in four rats.


Figure 3
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  		Figure 3: Transverse in vivo 1.5-T T2*-weighted gradient-echo MR images (16/2.8, 30° flip angle) of AAAs (in different animals) 7 (left), 14 (middle), and 28 (right) days after delivery of IONP VSMCs show areas of hypointense signal (arrows) around aortic wall. Vessel lumen appears as bright area (*). Scale bar = 1 mm.

 
All AAAs seeded with IONP VSMCs (in 10 rats) showed areas of hypointense signal at all time points. In nine of these rats, the initially circumferential area of hypointense signal became focal with time (Fig 4). One to four (mean, 3.0) foci of hypointense signal were detected on day 7; one to three (mean, 2.0) foci of hypointense signal, on day 14; and one to six (mean, 2.5) foci of hypointense signal, on day 28. Because of artifacts induced by iron-loaded cells, it was difficult to determine at 1.5-T MR imaging whether these areas of hypointense signal were localized in the neointima, media, or adventitia.


Figure 4
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  		Figure 4: Transverse T2*-weighted gradient-echo MR images (16/2.8, 30° flip angle) of AAAs immediately before (far left image) and days 0 (second image from left), 21 (third image from left), and 28 (far right image) after delivery of IONP VSMCs (in same animal) show areas of hypointense signal (arrows) in the aortic wall. Low-signal-intensity area is circular on day 0 and more focal (next to posterior part of aortic wall) on days 21 and 28. Vessel lumen appears as bright area (*). Scale bar = 1 mm.

 
Comparison of ex Vivo High-Field-Strength MR Imaging and Histologic Findings
MR imaging of the two AAAs harvested on day 28 performed ex vivo at 9.4 T revealed the areas of hypointense signal within the aortic wall that had been observed in vivo at 1.5-T MR imaging (Fig 5a). Perls Prussian blue–stained cross sections of the AAAs showed colocalization of the MR-depicted areas of hypointense signal and the iron-loaded cells deep within the neointima and in the adventitia (Fig 5b).


Figure 5A
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  		Figure 5a: (a) Transverse high-field-strength ex vivo 9.4-T T2*-weighted gradient-echo MR image (100/3, 25° flip angle) of aorta 28 days after delivery of IONP VSMCs shows crescent-shaped low-signal-intensity area (arrow) in deep portion of aortic wall, adjacent to a more focal low-signal-intensity area (black arrowhead). Focal nodular low-signal-intensity areas (white arrowhead) are also seen in external part of aortic wall. (b) Perls Prussian blue–stained histologic section (5 µm) of same area (original magnification, x40) shows iron-loaded cells in deep part of neointima (arrow and black arrowhead) and in adventitia (white arrowhead). Localization of iron-loaded cells is similar to that of low-signal-intensity areas in a. Scale bar = 0.5 mm.

 

Figure 5B
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  		Figure 5b: (a) Transverse high-field-strength ex vivo 9.4-T T2*-weighted gradient-echo MR image (100/3, 25° flip angle) of aorta 28 days after delivery of IONP VSMCs shows crescent-shaped low-signal-intensity area (arrow) in deep portion of aortic wall, adjacent to a more focal low-signal-intensity area (black arrowhead). Focal nodular low-signal-intensity areas (white arrowhead) are also seen in external part of aortic wall. (b) Perls Prussian blue–stained histologic section (5 µm) of same area (original magnification, x40) shows iron-loaded cells in deep part of neointima (arrow and black arrowhead) and in adventitia (white arrowhead). Localization of iron-loaded cells is similar to that of low-signal-intensity areas in a. Scale bar = 0.5 mm.

 
Comparison of in Vivo MR Imaging and Histologic Findings
Day 0.—The results of histologic analysis of AAAs at the time of seeding (day 0) confirmed the presence of a thrombus next to the aortic wall, as previously documented (5). Perls Prussian blue staining yielded findings negative for the detection of iron-loaded cells in both the AAAs seeded with non–magnetically labeled VSMCs (in three rats) and those injected with free iron oxide nanoparticles (in three rats). Sections of the AAAs seeded with IONP VSMCs showed iron-containing cells next to the aortic wall or within the thrombus on day 0 (Fig 6, A1). At this time point, all of the iron-loaded cells were positive for {alpha}-actin, a VSMC marker (Fig 6, B1). No macrophage detected in the AAAs (Fig 6, C1) showed iron loading.


Figure 6
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  		Figure 6: Histologic cross sections of AAAs on days 0–28 after VSMC delivery. A1–A4, Perls Prussian blue staining revealed iron-loaded cells (arrows) next to aortic wall on day 0 and in neointima on days 7, 14, and 28. C1–C4, Some Perls Prussian blue–negative macrophages (arrowheads) were detected on days 0, 7, 14, and 28. B1–B4, Colabeling for {alpha}-actin and Perls Prussian blue staining in the media revealed iron-containing cells positive for {alpha}-actin (arrows) on days 0, 7, 14, and 28. Some Perls Prussian blue–positive cells in the neointima and adventitia were negative at both macrophage staining (data not shown) and {alpha}-actin staining on days 14 and 28. Arrowheads in B3 and B4 indicate Perls Prussian blue–positive cells negative at {alpha}-actin staining in the neointima on days 14 and 28, respectively. Twin arrows in C3 indicate a Perls Prussian blue–positive cell in the adventitia on day 14. Some Perls Prussian blue–positive macrophages (arrow in C4) were also identified in the adventitia on day 28.

 
Follow-up.—The mean aneurysm diameters measured on T2-weighted spin-echo MR images and histologic cross sections after IONP VSMC seeding are reported in the Table. These measurements indicate a stability in AAA diameter after cell delivery (mean diameters at MR imaging on days 0 and 28: 3.1 and 2.7 mm, respectively), as previously observed after the seeding of nonlabeled VSMCs (5). Owing to the small sample size, no significant differences in diameter were noted.


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  		AAA Diameters Measured in Rats at in Vivo MR Imaging and Histologic Analysis

 
On days 7, 14, and 28, a neointima covered the luminal aspect of the media on the cross sections from all 10 rats that received IONP VSMC therapy. Perls Prussian blue staining revealed iron-containing cells in the neointima from day 7 to day 28 (Fig 6, A2–A4); the cells were usually localized in foci. On day 7, the iron-loaded cells were localized mainly in the middle portion of the neointima and less frequently in the adventitia. On days 14 and 28, the iron-loaded cells were localized mainly in the deep portion of the neointima (Fig 6, A3–A4).

We detected one to five (mean, 2.6) foci of iron-containing cells on day 7, one to two (mean, 1.5) such foci on day 14, and one to eight (mean, 3.0) such foci on day 28. The correlation between localization of low-signal-intensity areas in the aortic wall and repartition of iron-loaded cells in the aortic circumference was judged to be concordant in one (33%) of three rats on day 7, in two (67%) of three rats on day 14, and in two (50%) of four rats on day 28.

More than 80% of the iron-loaded cells in the neointima were positive for {alpha}-actin and negative for macrophage immunostain from day 7 to day 28; this finding suggests that these cells were generated from the seeded IONP VSMCs. Fewer than 20% of the iron-loaded cells in the neointima were negative for both {alpha}-actin and macrophage immunostain (Fig 6, second column). Several macrophages containing iron-labeled vesicles and a few cells negative for both {alpha}-actin and macrophage immunostain were also detected in the adventitia at late time points (Fig 6, C3–C4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
 References
 
Our study findings show that magnetically labeled VSMCs seeded into already formed AAAs in rats can be tracked in vivo by means of clinical MR imaging. We found that (a) magnetic cell labeling does not alter the therapeutic properties of VSMCs, (b) the topography and efficiency of cell delivery can be assessed at the time of the cell therapy procedure, and (c) many labeled cells detected at in vivo MR imaging remain confined to the regenerated area for up to 28 days after cell therapy.

The endovascular route of VSMC delivery was chosen because it is associated with low morbidity (3). In addition, VSMCs that develop on the luminal surface after cell therapy act as a platform for the delivery of paracrine factors to the aneurysmal wall (5,14,17). Although a relevant proportion of seeded cells may be washed away by aortic flow without associated clinical manifestations, some VSMCs adhere locally and proliferate to form a stabilizing intimal hyperplasia, as has been documented in other models (5,14,16,18).

T2*-weighted gradient-echo MR images depicted IONP VSMCs in all treated AAAs on day 0, the time point at which the iron-loaded cells were next to the aortic wall. The circular area of hypointense signal observed at in vivo MR imaging immediately after circumferential seeding reflected the topography of the cell deposition observed on the histologic cross sections. This was further documented by the MR depiction of cell seeding limited to one-quarter of the vessel circumference. Areas of hypointense signal were also apparent around the aortic wall and thus were suggestive of iron-loaded cell leakage around the aneurysm during cell delivery.

Low-signal-intensity areas were detected on 1.5-T T2*-weighted gradient-echo MR images throughout the time course of intimal development after cell seeding. Circular areas of hypointense signal observed on day 0 later became discontinuous and multifocal. The correlation between number and position of low-signal-intensity areas in the aortic wall circumference and foci of iron-loaded cells on histologic cross sections was poor after day 0, probably because of the low spatial resolution of clinical MR imaging of lesions 3 mm in diameter in this rodent model. Owing to high spatial resolution that approached histologic precision, 9.4-T MR imaging findings confirmed the excellent colocalization of low-signal-intensity areas with iron-loaded cells.

The mechanisms by which the initially circumferential areas of low signal intensity became patchy with time remain unclear. One could hypothesize that a substantial proportion of cells that were attached to the wall died shortly after delivery and were eliminated in the blood flow, so there was a discontinuous repartition of iron-loaded cells in the neointima.

We found that IONP VSMCs tended to be localized in the deepest area of the intimal hyperplasia; this finding suggests that these cells were covered up by a new layer of VSMCs. These VSMCs may have originated from the seeded cells after the cell divisions, from the recipient animal, or both. Double labeling revealed that the iron that was transferred during endovascular cell seeding was also located in a few cells that were negative for both VSMCs and macrophage immunostain and was located in both the neointima and the adventitia. These cells may have lost differentiation markers because of iron loading. However, in separate experiments, we demonstrated in vitro that the loading of VSMCs by means of iron oxide nanoparticle labeling did not modify the expression of {alpha}-actin (data not shown). In the adventitia, the presence of dedifferentiated cells could have resulted from the migration of IONP VSMCs after seeding or from an inadequate initial cell delivery (ie, leakage). The presence of iron-loaded macrophages in the adventitia suggests the uptake of iron nanoparticles and reflects the clearance of the tracer from the phagocytic system.

In vivo cell follow-up with MR imaging has been reported in other applications of cell therapy (19,20) with dextran-coated nanoparticles, which often requires long incubation times (24–48 hours) and the use of a transfection agent to achieve a sufficient iron load per cell. Anionic magnetic nanoparticles have the advantage of being rapidly internalized in vitro (2 hours) without use of a transfection agent (11,12,21). The short duration of incubation may facilitate the adoption of this technique for clinical applications.

In terms of study limitations, aortic thickening, a potential surrogate marker of disease correction (5), could not be depicted at MR imaging in the rodents. In humans, however, clinical MR imaging is a powerful tool used to document variations in aortic wall thickness (22), a potential marker of aortic healing (23).

In this study, precise localization of labeled cells in the aortic wall with 1.5-T MR imaging was limited by low spatial resolution. The aortic wall in rats is 250 µm (2 pixels) thick. The low spatial resolution could be overcome in rats by performing in vivo high-field-strength (>3 T) MR imaging or using new detection coils and imaging gradients (24). We also used a relatively short echo time, which can be increased to improve the detection of low-signal-intensity areas. In addition, other structures such as hemoglobin components may also induce low-signal-intensity areas at MR imaging, and a comparative analysis of images of the aorta before and after cell injection needs to be performed.

MR imaging combined with magnetic cell labeling may be an important tool in the development of endovascular cell therapy for AAAs, aimed toward clinical applications such as the endovascular treatment of endoleaks in humans. This is a first step toward the noninvasive evaluation of cell therapy for aortic aneurysms.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
 References
 


   IMPLICATION FOR PATIENT CARE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
References
 


   ACKNOWLEDGMENTS
 
The authors thank J. Bittoun, MD, PhD, for the MR experiments performed in the laboratory (Centre d'Imagerie et de Recherche en Résonance Magnétique Médicale, Le Kremlin-Bicètre, Paris, France), L. Louedec (INSERM U 698, Paris, France) and A. M. Guinault (CNRS Unité Nixte de Recherche 7054, Créteil, France) for excellent technical assistance, J. Prud'homme and D. Glutron for performing 1.5-T MR experiments (Centre d'Imagerie et de Recherche en Résonance Magnétique Médicale, Le Kremlin-Bicètre, Paris, France), F. Roudot-Thoraval for excellent statistical assistance, and A. Rahmouni and A. Luciani (Radiology Department, H. Mondor Hospital) for help editing the manuscript.


   FOOTNOTES
 

Abbreviations: AAA = abdominal aortic aneurysm • IONP = iron oxide nanoparticle labeled • VSMC = vascular smooth muscle cell

Guarantors of integrity of entire study, J.F.D., E.A.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, J.F.D., J.R., J.N.P., D.L., F.P.B., E.A.; experimental studies, J.F.D., J.D., C.R., F.G., P.M., B.G., J.R., J.N.P., D.L., E.A.; statistical analysis, E.A.; and manuscript editing, J.F.D., J.D., C.R., F.G., P.M., D.L., F.P.B., E.A.

Authors stated no financial relationship to disclose.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
IMPLICATION FOR PATIENT CARE
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