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

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).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   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.

View larger version (71K): [in this window] [in a new window] [Download PPT slide]   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.

View larger version (106K): [in this window] [in a new window] [Download PPT slide]   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.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   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.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   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.

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   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.

View larger version (90K): [in this window] [in a new window] [Download PPT slide]   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.

View larger version (106K): [in this window] [in a new window] [Download PPT slide]   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.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   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).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   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).

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   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).

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   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).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   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).

View larger version (91K): [in this window] [in a new window] [Download PPT slide]   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.