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DOI: 10.1148/radiol.2421051461
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(Radiology 2007;242:198-207.)
© RSNA, 2007

Molecular Imaging

Cell Therapy in Murine Atherosclerosis: In Vivo Imaging with High-Resolution Helical SPECT1

Sreekanth Vemulapalli, BS, Scott D. Metzler, PhD, Gamal Akabani, PhD, Neil A. Petry, MS, Nelsen J. Niehaus, MS, Xialin Liu, PhD, Nikhil H. Patil, BE, Kim L. Greer, CNMT, RT, Ronald J. Jaszczak, PhD, R. Edward Coleman, MD, Chunming Dong, MD, Pascal J. Goldschmidt-Clermont, MD and Bennett B. Chin, MD

1 From the Department of Medicine, Division of Cardiology (S.V., X.L., C.D., P.J.G.), and Department of Radiology (G.A., N.A.P., N.J.N., N.H.P., K.L.G., R.J.J., R.E.C., B.B.C.), Duke University Medical Center, Box 3808 DUMC, Durham, NC 27710; and Department of Radiology, University of Pennsylvania, Philadelphia, Pa (S.D.M.). Received September 1, 2005; revision requested November 3; revision received January 25, 2006; accepted February 6; final version accepted February 28. S.D.M. supported in part by National Institute for Biomedical Imaging and Bioengineering grants R01-EB-001910 and R33-EB-001543. R.J.J. supported in part by shared instrumentation funding from the National Center for Research Resources of the National Institutes of Health grant S10-RR-15697 and National Cancer Institute grant R01-CA-076006. S.V. supported in part by a Howard Hughes Medical Student Research Training Fellowship grant. Address correspondence to B.B.C. (e-mail: chin0004{at}mc.duke.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To determine the feasibility of in vivo localization and quantification of indium 111 (111In)-oxine–labeled bone marrow (BM) with high-resolution whole-body helical single photon emission computed tomography (SPECT) in an established murine model of atherosclerosis and vascular repair.

Materials and Methods: The institutional animal care and use committee approved this study. BM from young B6 Rosa 26 Lac Z+/+ mice was radiolabeled with 111In-oxine. On days 1, 4, and 7 after administration of radiolabeled cells, five C57/BL6 apolipoprotein E–deficient mice and five wild-type (WT) control mice were imaged with whole-body high-resolution helical SPECT. Quantification with SPECT was compared with ex vivo analysis by means of gamma counting. Autoradiography and ß-galactosidase staining were used to verify donor cell biodistribution. Linear regression was used to assess the correlation between continuous variables. Two-tailed Student t test was used to compare values between groups, and paired two-tailed t test was used to assess changes within subjects at different time points.

Results: SPECT image contrast was high, with clear visualization of BM, liver, and spleen 7 days after administration of radiolabeled cells. SPECT revealed that 42% and 58% more activity was localized to the aorta and BM (P < .05 for both), respectively, in apolipoprotein E–deficient mice versus WT mice. Furthermore, 28% and 27% less activity was localized to the liver and spleen (P < .05 for both), respectively, in apolipoprotein E–deficient mice versus WT mice. SPECT and organ gamma counts showed good quantitative correlation (r = 0.9). ß-Galactosidase staining and microautoradiography of recipient aortas showed donor cell localization to the intima of visible atherosclerotic plaque but not to unaffected regions of the vessel wall.

Conclusion: High-resolution in vivo helical pinhole SPECT can be used to monitor and quantify early biodistribution of 111In-oxine–labeled BM in a murine model of progenitor cell therapy for atherosclerosis.

© RSNA, 2007


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Data from animal and human studies have shown that deficiencies in bone marrow (BM)–derived progenitor cells are associated with atherosclerosis (1), risk factors for atherosclerosis (2), and endothelial dysfunction (3). Other studies have shown that BM-derived progenitor cells migrate and participate in neovascularization or vascular repair after ischemia or injury (4). In the well-established apolipoprotein E–deficient murine model of atherosclerosis (5), a therapeutic strategy to intravenously replace BM-derived progenitor cells has been shown to be effective in the prevention of atherosclerosis (1). In humans, levels of circulating endothelial progenitor cells correlate with endothelial function and are shown to have better value for predicting vascular reactivity than does the presence or absence of conventional risk factors (2). Furthermore, functional migration and proliferation capabilities of human circulating endothelial progenitor cells have been shown to be highly correlated with endothelial function (3).

The early stages of atherosclerosis result in the production and release of signaling cytokines, growth factors, and cellular adhesion molecules (6,7). After vascular injury, BM progenitor cells are recruited to facilitate vascular repair (4). It is hypothesized that recruitment of inflammatory cells from the BM represents a maladaptive process that occurs when the vascular progenitor cell population is insufficient for repair as a result of either depletion or senescence (8). This represents a new paradigm in the mechanism and treatment of atherosclerosis as a disease of impaired vascular repair that can be ameliorated with progenitor cell replacement. New strategies that involve the use of BM-derived progenitor cells to prevent or ameliorate vascular disease could greatly benefit from further characterization of factors that control mobilization, homing, engraftment, and differentiation of BM progenitor cells. Radionuclide imaging is a sensitive quantitative in vivo technique that may aid in determining how donor BM cells ameliorate or mitigate the development of atherosclerosis by identifying cell migration. In addition, radionuclide techniques that use indium 111 (111In)–oxine have been proved safe in humans; therefore, the translational potential for subsequent monitoring in human subjects is very high. Thus, the purpose of our study was to determine the feasibility of in vivo localization and quantification of 111In-oxine–labeled BM with high-resolution whole-body helical single photon emission computed tomography (SPECT) in an established murine model of atherosclerosis and vascular repair.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Animals
All procedures and studies were approved by the institutional animal care and use committee. Mice were purchased from Jackson Laboratory (Bar Harbor, Me). Five 20-week-old C57/BL6 apolipoprotein E–deficient mice on a high-fat atherogenic diet (study group) and five age-matched wild-type (WT) mice on a chow diet (control group) were anesthetized with ketamine (100 mg of ketamine per kilogram of body weight) and xylazine (10 mg/kg), and 111In-oxine–labeled donor whole BM was injected (S.V., X.L.) via the retro-orbital venous sinus. The apolipoprotein E–deficient mice were started on a high-fat diet (42% fat, 1.25% cholesterol) (#88137; Harlan-Teklad, Indianapolis, Ind) at 4 weeks of age. All apolipoprotein E–deficient mice and healthy control WT mice received between 9.5 x 106 and 1.42 x 107 radiolabeled B6 Rosa 26 Lac Z+/+ cells. Sequential imaging with high-resolution whole-body helical pinhole SPECT was performed in both groups 1, 4, and 7 days after injection.

In addition, mice in a second comparison group (three 20-week-old apolipoprotein E–deficient mice on a high-fat diet) were injected with 163–171 µCi of 111In-oxine (S.V., X.L.) via the retro-orbital sinus to determine the non–cell-bound 111In-oxine biodistribution. These mice underwent the same whole-body pinhole helical SPECT examination 1, 4, and 7 days after injection.

Dose Viability Testing of Lineage-Negative BM
Donor BM was isolated from young nonatherosclerotic control WT mice (S.V.). The femurs and tibias were flushed for BM with Dulbecco's modified Eagle medium (DMEM) (Invitrogen, Carlsbad, Calif) at 4°C. Whole BM was subsequently fractionated into lineage-positive and lineage-negative subfractions with magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Lineage-negative cells were resuspended in DMEM, counted, and separated into aliquots of approximately 4.75 x 104 cells (S.V.). Cells were radiolabeled by incubating each aliquot of lineage-negative cells with 0 µCi, 10 µCi, 100 µCi, or 500 µCi of 111In-oxine for 20 minutes at room temperature (S.V. N.A.P., N.J.N., B.B.C.) with a procedure that was similar to that used to radiolabel leukocytes (9). After incubation, cells were washed twice with DMEM and incubated in DMEM and 1.5% fetal bovine serum for 3 hours at 37°C and 5% CO2. The doses that were tested ranged (0.0–6.8) x 10–3 µCi of 111In-oxine per cell. Cell viability was determined after 3 hours of incubation with trypan blue staining (S.V., B.B.C.). BM cell radiolabeling efficiency was calculated by dividing the activity in cells by the total activity in cells and supernatants times 100. Results of these tests were used to estimate the 111In-oxine dose for subsequent cell radiolabeling in SPECT studies.

In Vitro BM Isolation and 111In-Oxine Radiolabeling
Donor BM was isolated from 6-week-old B6 Rosa 26 Lac Z+/+ donor mice (S.V.). The femurs and tibias were flushed for BM with DMEM at 4°C. Cells were resuspended in DMEM and passed through a 35-µm nylon filter (Becton Dickinson, Franklin Lakes, NJ). For 111In-oxine radiolabeling, (5 to 6) x 107 cells were incubated with 1.6 mCi of 111In-oxine (27–32 µCi of 111In-oxine per 106 cells) for 20 minutes at room temperature (S.V., B.B.C., N.A.P., N.J.N.). Radiolabled cells were then washed twice in DMEM and divided into aliquots of approximately 107 cells. After incubation, cell viability and radiolabeling efficiency were measured at 1 hour, as stated previously. The final administered dose of 111In-oxine–radiolabeled cells ranged from approximately 10 to 14 µCi of 111In-oxine per 106 cells.

Technetium 99m Red Blood Cell Labeling
To enable anatomic localization of activity to the aorta, the in vivo technetium 99m (99mTc) red blood cell radiolabeling method used in humans was modified, tested, and validated in mice (S.V., B.B.C., N.A.P., N.J.N.) (10). Briefly, 4 days after donor cell injection, all apolipoprotein E–deficient mice and control mice received an injection of 0.4 µg of stannous pyrophosphate. After 25–40 minutes, they received an injection of 1.15–1.87 mCi of 99mTc pertechnetate, which resulted in more than 95% radiolabeling efficiency when measured at 1 hour. All mice then underwent dual-isotope 99mTc and 111In-oxine SPECT.

In Vivo High-Resolution Helical SPECT
Whole-body high-resolution SPECT (B.B.C., S.D.M., N.H.P., R.J.J., S.V.) was serially performed with a helical pinhole SPECT acquisition technique in all mice 1, 4, and 7 days after administration of donor cells (11). Pinhole SPECT images were acquired with specially designed pyramidal collimators with 1.0-mm pinhole tungsten apertures and additional shielding for the medium-energy radionuclide 111In-oxine (11). A small radius of rotation (28 mm) was used to achieve high magnification (x7.4) and high spatial resolution. A triple-detector gamma camera (Triad XLT; Trionix, Twinsburg, Ohio) was used to acquire two photopeaks for 111In-oxine imaging (171 KeV [5% energy window] and 245 KeV [10% energy window]) with the following parameters: step and shoot rotation, image acquisition time of 15 seconds per view, 180 images acquired, 2° step in 360° rotation, and 28-mm radius of rotation. On day 7, image acquisition time was doubled to 30 seconds per view to partially compensate for radiotracer decay. Dual-isotope imaging (111In-oxine imaging as described previously and 99mTc imaging at 140 KeV [10% energy window]) was also performed on day 4 after 99mTc red blood cell radiolabeling for vascular blood pool localization. With use of a narrow lower-energy window setting for 111In-oxine photopeak imaging and a relatively low dose of 99mTc, dual-isotope imaging did not show detectable cardiac blood pool activity in the 111In-oxine photopeak.

Helical SPECT images were acquired by means of linear translocation of mice through the axis of rotation with a computer-controlled linear stage (12). This enabled complete data sampling of the whole body to be performed in a single examination. Images were acquired with a 256 x 128 matrix and a reconstructed voxel size of 0.125 mm3. SPECT images were iteratively reconstructed by using an ordered subset expectation maximization algorithm with four subsets (13,14). Images were displayed and region-of-interest (ROI) analysis was performed with customized software (Specter 4.0; Duke University, Durham, NC). The dual-isotope images were displayed and analyzed with the GNU Image Manipulation Program, version 1.2 (Free Software Foundation, Boston, Mass).

Biodistribution of activity was determined with ROI analysis of SPECT images: ROIs were drawn around all visible organs, including the liver, spleen, lungs, kidneys, and aorta (S.V.). ROIs were also drawn in the cardiac blood pool on both the 111In-oxine cell-bound and the 111In-oxine non–cell-bound images to quantify blood pool activity. Because BM activity was difficult to trace in all sections, it was calculated by drawing an ROI around the body in the transverse section, subtracting the activity in included organs (ie, lungs and kidneys), and adding all transverse sections with BM activity. All counts were corrected for imaging time and radiotracer decay. These results were compared with decay-corrected gamma counts in explanted organs.

Gamma Counting in Explanted Organs
Gamma counts were obtained in explanted organs and compared with aortic activity on SPECT images to determine if there was a correlation (S.V. B.B.C., N.H.P., N.J.N.). All mice were humanely euthanized 8 days after administration of donor cells. The aorta, spleen, liver, quadriceps muscle, kidneys, and lungs were dissected, weighed, and counted in a gamma well counter (Autogamma 5000 Series; Packard, Meriden, Conn) according to a standardized protocol. This included isotope-specific correction for decay, background, gamma counter efficiency, and conversion from counts per minute to microcuries. Activity in each organ was expressed as a percentage of total activity administered.

Microautoradiography and ß-Galactosidase Staining
Microautoradiography and ß-galactosidase staining were used to confirm the presence of 111In-oxine in donor cells and to further investigate the localization of donor cells at histologic analysis (S.V. G.A.). Explanted organs were preserved in 30% sucrose and frozen in an embedding compound (Tissue Tek; Sakura, Torrance, Calif). Thereafter, 15-µm frozen slices of the spleens, livers, and aortas of the imaged mice were cut and fixed for 10 seconds in 10% formalin and coated in LM-1 microautoradiographic emulsion film (Amersham, Buckinghamshire, England) in a dark room. Tissue slices were exposed to microautoradiographic emulsion for 3 weeks at 4°C and then developed by using standard developer (Kodak D-19; Eastman Kodak, Rochester, NY) before they were viewed with light microscopy. The initial frozen sections were stained and assayed for ß-galactosidase activity before microautoradiographs were obtained. Sections were incubated with 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-Gal; American Bioanalytical, Natick, Mass) in buffer at 37°C for 90 minutes and dehydrated for viewing with light microscopy.

Statistical Analysis
Data are reported as means ± standard deviations (S.V., B.B.C.). Findings in atherosclerotic apolipoprotein E–deficient mice were compared with findings in control WT mice by using an unpaired two-tailed Student t test. Comparisons in the same mice over time were performed with a paired two-tailed t test. P values of less than .05 were considered to indicate statistical significance. Linear regression with the least-squares method was used to compare the continuous variables of organ activity measurements with gamma well counter measurements and SPECT findings. A no-intercept model was used to compare SPECT findings with gamma well counter measurements. This model incorporated an assumption of negligible counts with SPECT in the absence of activity and zero gamma well counter results with routine background subtraction calibration.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
In Vitro BM Radiolabeling with 111In-Oxine
No immediate viability change was seen after 3 hours of cell incubation until the concentration reached at least 1653 µCi of 111In-oxine per 106 cells (Fig 1). Subsequent 111In-oxine cell radiolabeling of BM for imaging studies was performed with radioactivity concentrations that were approximately 50–60 times lower. Doses of 27–32 µCi of 111In-oxine per 106 cells correspond to the 1.6-mCi dose of 111In-oxine used to radiolabel (5 to 6) x 107 BM cells. For in vivo imaging experiments, radiolabeling efficiency of 111In-oxine in whole BM cells was 59.0% ± 3.5 and cell viability 1 hour after radiolabeling was 93.7% ± 7.6.


Figure 1
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  		Figure 1: Graph shows 3-hour viability of 111In-oxine–labeled lineage-negative BM. Lineage-negative BM cells from radiolabeled B6 Rosa 26 Lac Z+/+ cells were incubated with varying concentrations of 111In-oxine and assayed for viability after 3 hours. Each point represents the average of two independent experiments. Doses used for in vivo imaging experiments were well below the lowest doses that affect cell viability.

 
Donor BM Localization
SPECT images obtained on day 1 showed migration of donor BM to the liver, spleen, and BM of the femur, tibia, pelvis, and lumbar spine (Fig 2, A–D) in all mice in both the apolipoprotein E–deficient group and the control group. Readily identifiable organs of donor BM migration were also noted at 7 days in all mice. While migration of donor BM to the liver, spleen, and BM of the femur, tibia, pelvis, and lumbar spine was evident in both groups 1 day after cell administration, donor BM localization to the aorta occurred only in apolipoprotein E–deficient mice (Fig 2, E–H). Intensity profiles of the thoracic spine and aorta (Fig 2, G, H) show that SPECT enabled differentiation of the spine and aorta.


Figure 2
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  		Figure 2: Donor cells migrate preferentially to the aorta in atherosclerotic apolipoprotein E–deficient mice (B6 ApoE–/–) as compared with nonatherosclerotic control mice (B6 WT). A, C, anterior, and, B, D, left lateral reprojection SPECT images obtained 1 day after retro-orbital administration of Rosa 26 Lac Z+/+ radiolabeled donor cells. E, F, Thoracic transaxial SPECT images obtained just below the level of the aortic arch 1 day after administration of radiolabeled donor BM cells. G, Intensity profile along the dotted line in E indicates donor cell localization to both the spine and the aorta in atherosclerotic apolipoprotein E–deficient mice. H, Intensity profile along the dotted line in F indicates donor cell localization to the spine in only nonatherosclerotic control mice. Hot iron color scale indicates 111In-oxine activity. L = liver, S = spleen.

 
For all mice that received only 111In-oxine without BM (n = 3), the biodistribution of non–donor-cell-bound 111In-oxine in the blood pool and soft-tissue background was visually apparent and distinct from that of 111In-oxine–labeled BM cell activity. At 1 day after radionuclide administration, prominent blood pool activity was visually apparent in the heart, kidneys, liver, and spleen of all mice; prominent soft-tissue activity was also present and persisted through imaging at day 7 (Fig 3, A–F). Non–donor-cell-bound 111In-oxine showed 1.31% ± 0.06 activity quantified with SPECT in the transaxial section of the cardiac blood pool on day 4, which was more than 10 times higher than the corresponding 0.09% ± 0.02 activity seen in the aorta in these mice. In all apolipoprotein E–deficient mice that received donor BM, cardiac blood pool activity was visually compared with aortic activity during the 111In-oxine photopeak. In all cases, faint aortic activity was visible without evidence of cardiac blood pool activity. The absence of 111In-oxine activity in the cardiac ventricular blood pool also provided further evidence that enabled us to confirm that activity seen in the aorta was localized to the aortic wall and was not in the blood pool.


Figure 3
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  		Figure 3: A–C, Anterior, and, D–F, corresponding left lateral reprojection SPECT images in a representative B6 apolipoprotein E–deficient mouse show non–donor-cell-bound 111In-oxine localizes to the blood pool (BP) and soft tissue. G, Thoracic transaxial SPECT image obtained at the left midventricular level in the same mouse demonstrates blood pool anatomy and enables comparison with BM. H, Sagittal, and, I, coronal SPECT images obtained at day 1 in the same mouse. In the three non–donor-cell-bound mice, higher background and blood pool activity were present on all images when compared with 111In-oxine BM images. Hot iron color scale indicates 111In-oxine activity. K = kidney, L = liver, LV = left ventricle, RV = right ventricle.

 
Aortic Donor Cell Localization with 99mTc Red Blood Cell and 111In-Oxine Dual-Isotope Imaging
Images of the 111In-oxine photopeak demonstrated greater activity in the aorta of atherosclerotic apolipoprotein E–deficient mice than in the aorta of nonatherosclerotic control mice (Fig 4). In all five apolipoprotein E–deficient BM recipient mice, 111In-oxine images showed no activity in the cardiac ventricular blood pool.


Figure 4
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  		Figure 4: SPECT images obtained in, A–F, an atherosclerotic B6 apolipoprotein E–deficient mouse (B6 ApoE–/–), and, G–L, a nonatherosclerotic control mouse (B6 WT) 4 days after retro-orbital administration of 111In-oxine radiolabeled donor cells. Sagittal A, 111In-oxine–labeled, B, fusion, and, C, concurrent 99mTc-labeled red blood cell images show delineation of donor BM activity biodistribution compared with that in the blood pool (BP). Transaxial D, 99mTc-labeled blood pool, E, fusion, and, F, 111In-oxine–labeled SPECT images show colocalization of BM donor activity in the aorta. This finding is in contrast to findings in control mice; fusion images in control mice do not demonstrate activity in the aorta. Hot iron color scale indicates 111In-oxine activity; black and white color scale indicates 99mTc activity.

 
ROI analysis of SPECT images obtained on day 4 (Fig 5a) enabled us to confirm that donor cells were localized preferentially in the aorta in atherosclerotic apolipoprotein E–deficient mice versus nonatherosclerotic WT control mice (0.027% ± 0.001 vs 0.019% ± 0.004, P = .01). The aortic activity, as determined with SPECT images obtained on day 4, and gamma counts of aortas explanted on day 8 (Fig 5b) showed good correlation (r = 0.82).


Figure 5
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  		Figure 5a: Graphs show correlation between donor cell aortic activity localization with SPECT and with gamma well counting. (a) Donor cells localize preferentially to the recipient aorta, as determined with SPECT (P = .01) and confirmed with gamma well counting of explanted recipient aortas (P = .01). (b) A good linear correlation (r = 0.82) exists between the percentage of aortic activity determined with SPECT and the percentage of aortic activity determined with gamma counting. ApoE–/– = apolipoprotein E–deficient mouse, WT = nonatherosclerotic control mouse.

 

Figure 5
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  		Figure 5b: Graphs show correlation between donor cell aortic activity localization with SPECT and with gamma well counting. (a) Donor cells localize preferentially to the recipient aorta, as determined with SPECT (P = .01) and confirmed with gamma well counting of explanted recipient aortas (P = .01). (b) A good linear correlation (r = 0.82) exists between the percentage of aortic activity determined with SPECT and the percentage of aortic activity determined with gamma counting. ApoE–/– = apolipoprotein E–deficient mouse, WT = nonatherosclerotic control mouse.

 
111In-Oxine–labeled Donor Cell Localization to Aortic Plaque
Microautoradiography and X-Gal staining for ß-galactosidase activity on frozen aortic sections showed intracytoplasmic accumulation of 111In-oxine activity and ß-galactosidase within intimal cells of aortic plaques but not within the intima of unaffected regions within the same aortic section. Coregistration of ß-galactosidase activity and 111In-oxine activity showed localization within cells that morphologically resembled endothelial cells (Fig 6).


Figure 6
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  		Figure 6: Microautoradiographs show ß-galactosidase and 111In-oxine–labeled donor cells in the aorta and spleen of an atherosclerotic apolipoprotein E–deficient mouse. A, X-gal staining of a transverse frozen section of the aortic arch shows donor cells preferentially localized to the intima of atherosclerotic plaques (arrows). B, High-power magnification of healthy intima seen in A. C, High-power magnification of donor cells localizing to atherosclerotic plaques seen in A. D, Frozen slice of the spleen of a B6 apolipoprotein E–deficient mouse stained with ß-galactosidase after autoradiography to localize 111In-oxine. ß-Galactosidase–positive 111In-oxine–negative cells (top arrows) appear bluer than ß-galactosidase–positive 111In-oxine–positive cells (bottom arrow). The blue stain is an indicator of ß-galactosidase activity from donor BM.

 
Donor Whole BM Localization
Images obtained 1 day after administration of donor whole BM in apolipoprotein E–deficient mice and nonatherosclerotic control mice showed similar patterns of extraaortic migration to the liver, spleen, and BM. By day 7, however, localization to the BM in nonatherosclerotic control mice was substantially diminished compared with that in atherosclerotic apolipoprotein E–deficient mice (Fig 7). ROI analysis of SPECT images obtained at day 7 (Fig 8a) revealed that activity was localized preferentially to the BM in the atherosclerotic apolipoprotein E–deficient mice versus activity in the WT control mice (39.6% ± 8.7 vs 25.0% ± 6.4, P = .02). Activity in the liver (44.9% ± 5.3 vs 35.0% ± 6.2, P = .03) and spleen (19.0% ± 2.7 vs 15.0% ± 2.3, P = .04) was higher in control mice than in apolipoprotein E–deficient mice.


Figure 7
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  		Figure 7: Representative anterior and left lateral reprojection SPECT images in, A, B, an apolipoprotein E–deficient (B6 ApoE–/–) mouse, and, C, D, a control (B6 WT) mouse 7 days after donor cell administration show donor cell activity preferentially localizes to recipient BM in atherosclerotic apolipoprotein E–deficient mice. L = liver, S = spleen. Hot iron color scale indicates 111In-oxine activity.

 

Figure 8
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  		Figure 8a: (a) Graph shows 111In-oxine–labeled donor cell localization in recipient apolipoprotein E–deficient atherosclerotic mice (ApoE–/–) versus that in WT control mice, as determined with whole-body SPECT at 7 days. Localization to BM was significantly (P = .02) higher in apolipoprotein E–deficient mice than in control mice. In contrast, localization to the spleen and liver (P = .04 and P = .02, respectively) was significantly lower in apolipoprotein E–deficient mice than in control mice. (b, c) Graphs show linear correlation for whole-body 111In-oxine activity per organ, as determined with gamma counts and SPECT in apolipoprotein E–deficient (b) and control (c) mice. Activity determined with SPECT shows good correlation with activity determined with gamma well counting for apolipoprotein E–deficient and control mice (r = 0.92 and = 0.91, respectively).

 
Regarding the comparison of day 7 SPECT quantification with gamma well counts of explanted organs (Figs 8b, 8c), the least-squares correlation coefficients for B6 apolipoprotein E–deficient mice and B6 control mice were 0.92 and 0.91, respectively; this indicated a linear relationship between SPECT counts and gamma well counts.


Figure 8
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  		Figure 8b: (a) Graph shows 111In-oxine–labeled donor cell localization in recipient apolipoprotein E–deficient atherosclerotic mice (ApoE–/–) versus that in WT control mice, as determined with whole-body SPECT at 7 days. Localization to BM was significantly (P = .02) higher in apolipoprotein E–deficient mice than in control mice. In contrast, localization to the spleen and liver (P = .04 and P = .02, respectively) was significantly lower in apolipoprotein E–deficient mice than in control mice. (b, c) Graphs show linear correlation for whole-body 111In-oxine activity per organ, as determined with gamma counts and SPECT in apolipoprotein E–deficient (b) and control (c) mice. Activity determined with SPECT shows good correlation with activity determined with gamma well counting for apolipoprotein E–deficient and control mice (r = 0.92 and = 0.91, respectively).

 

Figure 8
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  		Figure 8c: (a) Graph shows 111In-oxine–labeled donor cell localization in recipient apolipoprotein E–deficient atherosclerotic mice (ApoE–/–) versus that in WT control mice, as determined with whole-body SPECT at 7 days. Localization to BM was significantly (P = .02) higher in apolipoprotein E–deficient mice than in control mice. In contrast, localization to the spleen and liver (P = .04 and P = .02, respectively) was significantly lower in apolipoprotein E–deficient mice than in control mice. (b, c) Graphs show linear correlation for whole-body 111In-oxine activity per organ, as determined with gamma counts and SPECT in apolipoprotein E–deficient (b) and control (c) mice. Activity determined with SPECT shows good correlation with activity determined with gamma well counting for apolipoprotein E–deficient and control mice (r = 0.92 and = 0.91, respectively).

 
Although there was no significant change (Fig 9) in biodistribution over time on days 1, 4, and 7 in apolipoprotein E–deficient mice, the control mice showed a significant decrease in activity in the BM from day 1 to day 7 (35.8% ± 10.8 vs 25.0% ± 6.4, P = .02) and a concomitant increase in localization to the liver (39.4% ± 8.4 vs 44.9% ± 5.3, P = .03). Thus, there was an initial donor biodistribution difference in the aorta, as well as a later differential migration from the BM to the liver in the nonatherosclerotic control mice (Fig 10).


Figure 9
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  		Figure 9a: (a, b) Graphs show donor cell activity migrates from the BM to the liver in control mice (b) but not in atherosclerotic apolipoprotein E–deficient mice (a). Percentages of whole-body activity, as determined with SPECT, are plotted against time after donor cell administration. In a, there is no significant difference in mean BM activity (43.5% vs 39.4%, P = .35) or mean liver activity (31.3% vs 34.1%, P = .13) between days 1 and 7. In b, there is a significant decrease in BM localization (35.8% vs 27.0%, P = .02) and an increase in liver localization (39.4% vs 44.9%, P = .03) between days 1 and 7, which indicate a net migration of donor cells between these organs. Mean spleen activity in control mice increased from day 1 to day 7; however, the increase was not significant (14.1% vs 19.0%, P = .08).

 

Figure 9
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  		Figure 9b: (a, b) Graphs show donor cell activity migrates from the BM to the liver in control mice (b) but not in atherosclerotic apolipoprotein E–deficient mice (a). Percentages of whole-body activity, as determined with SPECT, are plotted against time after donor cell administration. In a, there is no significant difference in mean BM activity (43.5% vs 39.4%, P = .35) or mean liver activity (31.3% vs 34.1%, P = .13) between days 1 and 7. In b, there is a significant decrease in BM localization (35.8% vs 27.0%, P = .02) and an increase in liver localization (39.4% vs 44.9%, P = .03) between days 1 and 7, which indicate a net migration of donor cells between these organs. Mean spleen activity in control mice increased from day 1 to day 7; however, the increase was not significant (14.1% vs 19.0%, P = .08).

 

Figure 10
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  		Figure 10: Reprojection SPECT images of, A, B, an atherosclerotic apolipoprotein E–deficient mouse and, C, D, a nonatherosclerotic WT control mouse obtained 1 day (A and C) and 7 days (B and D) after 111In-oxine–labeled donor cell administration. Donor cell activity migrates from the BM to the recipient liver in control mice but not in atherosclerotic apolipoprotein E–deficient mice. Differences in cell biodistribution are apparent 7 days after donor cell administration; images in the control mouse demonstrate lower BM activity and higher liver and spleen activity at day 7 than do images in the apolipoprotein E–deficient mouse.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
To our knowledge, our study is the first in which radiolabeled BM cells were depicted with high-resolution whole-body SPECT in the context of a murine model of cell therapy for atherosclerosis. Specifically, 0.027% of intravenously administered donor cells were retained in atherosclerotic aortas; this was an increase of 140% over that seen in unaffected aortas. We observed an early localization (within 4 days of administration) of donor cells to apolipoprotein E–deficient recipient aortas. This early localization specifically to sites of atherosclerotic injury represents new evidence that vascular injury signals the early homing of donor cells specifically to these sites of injury.

Microautoradiography and X-gal staining of atherosclerotic aortas revealed cell-associated 111In-oxine activity and ß-galactosidase activity preferentially in areas of intimal plaque. Engraftment of donor cells demonstrated a morphology of endothelial cells within intimal plaques in atherosclerotic aortas. This is consistent with the findings of a previous study that showed that donor cells localize to the aorta in a distribution that is concordant with the localization of atherosclerotic plaques (1). At present, the precise cell type(s) that is responsible for mitigating atherosclerosis in this murine model is unknown. Administration of whole BM from young syngeneic mice, which includes progenitor cells, has been the only cellular therapy to lead to the effective prevention of atherosclerosis in this animal model (1). The predominant cell phenotype (62% of cells) that demonstrates homing to the atherosclerotic lesions is in the CD31+/CD45– population, which is consistent with a cell population of endothelial origin (15). The phenotype that includes macrophages (CD31–/CD45+) was also present but was found to occur in a smaller percentage (22%) of cells (1). Although more than one cell population may be responsible for mitigation of atherosclerosis, these data support the hypothesis that endothelial progenitor cells play an important role in this process.

We also identified and quantified a preferential localization of activity in the BM of the apolipoprotein E–deficient atherosclerotic mice. This localization was significantly higher (P = .02) than that in control mice by day 7. The control mice showed a significant decrease in the activity in the BM from day 1 to day 7 (P = .02). These data support the hypothesis that exogenous donor cell administration mitigates atherosclerosis in part by replenishing the depleted or functionally impaired BM progenitor cell population in atherosclerotic mice. These data are also in agreement with previous data that show atherosclerotic apolipoprotein E–deficient mice have a depleted population of CD31+/CD45– intermediate progenitor cells in the BM compared with control mice (1). In studies involving humans, the loss of endothelial progenitor cells in the peripheral circulation, either in absolute numbers or in function, is also associated with increased Framingham risk score (2) and endothelial dysfunction (3).

From an imaging perspective, high-spatial-resolution and high-sensitivity whole-body SPECT with helical acquisitions is now possible by means of advanced instrumentation and reconstruction algorithms (11,12,16,17). 111In-oxine radiolabeling and helical whole-body pinhole SPECT have enabled the quantitative evaluation of donor activity biodistribution to the BM and other organs. Gamma counting in explanted organs was used to verify quantitative accuracy, and microautoradiography with X-gal staining was used to verify cell migration. Radionuclide imaging is an efficient and accurate method for localization and quantification of activity, which normally are difficult logistical tasks, especially in regions such as the BM. Traditional ex vivo histologic methods used to quantify cells in BM entail estimations based on assumptions and representative sampling. Other imaging methods, such as magnetic resonance (MR) imaging, have high spatial resolution but are similarly limited in quantitative capability.

Radiolabeling of BM with 111In-oxine may be especially advantageous for cell migration applications. 111In-oxine is lipophilic and passively diffuses into cells, where it dissociates and subsequently binds with high affinity to intracytoplasmic proteins (9). Thus, it may be used to radiolabel a wide variety of cells, irrespective of cell surface membrane expression or metabolic state. Although there are other methods related to viral vector transfection for cell migration, our method avoids the issues of altering cellular genetics, thus producing consistent gene expression and avoiding potential for immunologic reactions. The use of 111In-oxine also simplifies the technical logistics of experimental design by reducing the need for special animal isolation facilities for viral vectors and dedicated radiotracer synthesis.

Potential disadvantages of using 111In-oxine exist, and they are currently being investigated. The long-term viability and function of radiolabeled donor cells were not assessed. In the group of apolipoprotein E–deficient mice, donor cell migration to sites of atherosclerosis and BM was significantly different from that in the control group. This finding provides evidence to support functional preservation of cell migration. Non–cell-bound 111In-oxine studies also revealed a high level of soft-tissue activity that was not revealed by the radiolabeled donor cell studies. Our in vitro studies also demonstrated no loss of cell viability until doses approximately 50 times higher than those used in this study were used.

Previous study investigators have used 111In-oxine to radiolabel and quantify human endothelial progenitor cells (18,19). These studies, however, involved the use of planar imaging in larger rat models of myocardial infarction and different cell labeling techniques. Although higher concentrations of 111In-oxine led to impairment of cell viability and function, we and others (20) have found that a relatively low dose of 111In-oxine does not affect the immediate viability of BM or the lineage-negative fraction. In addition, radiolabeling does not affect endothelial progenitor cell proliferation and migration as adversely as it affects the less mature hematopoietic progenitor cells (19). Finally, in our study we used freshly isolated BM cells, whereas previous study investigators used thawed cells that had been isolated for several years before viability testing (19).

MR imaging has also shown great promise as a noninvasive modality for monitoring cell therapy (21–25). In our model of atherosclerosis, however, sequential imaging and whole-body quantification are important because the donor cell biodistribution after intravenous administration is unknown. As the field of progenitor cell therapy evolves, further studies in more specifically identified donor cell populations are likely to enhance our understanding of the mechanisms underlying the dramatic effects of progenitor cell therapies.

To our knowledge, this is the first study to reveal the ability of high-resolution whole-body helical SPECT to aid in the localization and quantification of the in vivo biodistribution of radiolabeled BM. This modality has the potential to be used to study cellular therapies for atherosclerosis in mice. Quantitative data demonstrated preferential homing of cells to the aorta and BM in atherosclerotic mice, which supports the hypothesis that atherosclerosis is a process of chronic injury and that the therapeutic effect of donor BM cells is a product of direct migration of cells to the aorta and replenishment of depleted or functionally senescent vascular progenitor cells. Helical high-resolution SPECT is a feasible method that can be used to identify and quantify radiolabeled cells in cellular therapies for atherosclerosis in murine models. Because 111In-oxine is approved for human use, the findings of this study have high translational potential for future studies of human cellular therapies.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   FOOTNOTES
 

Abbreviations: BM = bone marrow • DMEM = Dulbecco's modified Eagle medium • ROI = region of interest • WT = wild type

Author contributions: Guarantors of integrity of entire study, S.V., G.A., P.J.G., B.B.C.; 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, S.V., G.A., X.L., C.D., P.J.G., B.B.C.; experimental studies, S.V., S.D.M., G.A., N.A.P., N.J.N., X.L., N.H.P., K.L.G., R.E.C., C.D., B.B.C.; statistical analysis, S.V., S.D.M., G.A., B.B.C.; and manuscript editing, S.V., S.D.M., G.A., N.A.P., K.L.G., R.E.C., C.D., P.J.G., B.B.C.

Authors stated no financial relationship to disclose.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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