In Vivo Molecular Imaging of Atherosclerotic Lesions in ApoE−/− Mice Using VCAM-1–Specific, 99mTc-Labeled Peptidic Sequences

Julien Dimastromatteo; Alexis Broisat; Pascale Perret; Mitra Ahmadi; Didier Boturyn; Pascal Dumy; Daniel Fagret; Laurent M. Riou; Catherine Ghezzi

doi:10.2967/jnumed.112.115675

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FIGURE 1.
Histologic (hematoxylin, erythrosine, and safran) and immunohistologic staining of VCAM-1 and Mac-2 expression in ApoE^−/− mouse carotid vessels. Histochemistry and immunohistochemistry were performed on adjacent transverse sections of atherosclerotic lesions from ligated left carotid arteries (A–E), with magnifications centered on intimal and adventitial areas (F–J and K–O, respectively) and on nonligated left carotid arteries from sham-operated ApoE^−/− animals (P–T) and on contralateral, right, carotid arteries (U–Y). Positive VCAM-1 and Mac-2 immunostaining was observed in atherosclerotic lesions developing at site of left carotid artery ligation but not in vessels from sham-operated animals and right carotid arteries. Specificity of immunostaining was assessed by performing control experiments in absence of VCAM-1– and Mac-2–specific primary antibodies (without VCAM-1 Ab and without MAC-2 Ab). Ab = antibody; HES = hematoxylin, erythrosine, and safran; w/o = without.*Vessel lumen.
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FIGURE 2.
Left-to-right carotid (A) and left carotid-to-blood (B) activity ratios of ^99mTc-labeled peptidic sequences as determined by γ-well counting at 180 min after intravenous tracer injection. mis. = mismatch. *P < 0.05 vs. ^99mTc-B2702p. ^†P < 0.05 vs. ^99mTc-B2702p1 mismatch.
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FIGURE 3.
Time–activity curves of ^99mTc-B2702p1 in blood and left and right carotid arteries at 15, 60, and 180 min after intravenous injection of tracer.
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FIGURE 4.
(A) Representative planar images of ApoE^−/− mouse with left carotid artery ligation at 180 min after injection of ^99mTc-B2702p1, ^99mTc-B2702p1 mismatch, and ^99mTc-B2702p; corresponding left-to-right carotid tracer activity ratios are indicated in parentheses. (B) ^99mTc-B2702p1, ^99mTc-B2702p1 mismatch, and ^99mTc-B2702p left-to-right carotid activity ratios from in vivo planar image quantification. *P < 0.05 vs. ^99mTc-B2702p. ^†P < 0.05 vs. ^99mTc-B2702p1 mismatch. mis = mismatch.
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FIGURE 5.
(A) Representative pinhole SPECT image of ^99mTc-B2702p1 and ^99mTc-B2702p activity at level of atherosclerotic lesion developing at site of left carotid artery ligation in ApoE^−/− mouse. Corresponding left-to-right carotid tracer activity ratios are indicated in parentheses. (B) Left and right ^99mTc-B2702p1 (left) and ^99mTc-B2702p (right) carotid activities from in vivo pinhole SPECT image quantification. *P < 0.05 vs. right carotid tracer activity.
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FIGURE 6.
Anisotropy values from [F]-B2702p1 in presence of increasing concentrations of recombinant human VCAM-1. Results from control experiments performed in absence of VCAM-1 and in presence of nonspecific target bovine serum albumin are shown in inset.

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TABLE 1

Peptidic Sequence, Radiochemical Purity, and Urinary Stability of ^99mTc-B2702p1-20 Peptides

Tracer	Peptidic sequence	RCP (%)	Stability (%)
^99mTc-B2702p	H₂N-HGR ENL RIA LRY-COOH	90	93
^99mTc-B2702p1	H₂N-HGR ANL RIL ARY-COOH	92	94
^99mTc-B2702p1 mismatch	H2N- HGL RAY IRA RNL-COOH	90	89
^99mTc-B2702p2	H₂N-HGR ENL AIL ARY-COOH	95	96
^99mTc-B2702p3	H₂N-HGR ENL RIL ARA-COOH	97	83
^99mTc-B2702p4	H₂N-HGR ENL RIL AAY-COOH	95	92
^99mTc-B2702p5	H₂N-HGR ENL RIL ARY-COOH	50	N/D
^99mTc-B2702p6	H₂N-HGR ENA RIL ARY-COOH	93	95
^99mTc-B2702p7	H₂N-HGA ENL RIL ARY-COOH	95	50
^99mTc-B2702p8	H₂N-HGR ENL RIA ARY-COOH	80	84
^99mTc-B2702p9	H₂N-HGR EAL RIL ARY-COOH	95	97
^99mTc-B2702p10	H₂N-HGR ENL RIL ARY-COOH	95	99
^99mTc-B2702p11	H₂N-HGA ENL RIA LRY-COOH	97	82
^99mTc-B2702p12	H₂N-HGR ANL RIA LRY-COOH	88	80
^99mTc-B2702p13	H₂N-HGR EAL RIA LRY-COOH	96	79
^99mTc-B2702p14	H₂N-HGR ENA RIA LRY-COOH	96	76
^99mTc-B2702p15	H₂N-HGR ENL AIA LRY-COOH	96	85
^99mTc-B2702p16	H₂N-HGR ENL RAA LRY-COOH	91	70
^99mTc-B2702p17	H₂N-HGR ENL RIA LAY-COOH	90	80
^99mTc-B2702p18	H₂N-HGR ENL RIA LRA-COOH	88	81
^99mTc-B2702p19	H₂N-HGR ANL RIL ARA-COOH	88	79
^99mTc-B2702p20	H₂N-HGR ANL RIL AAY-COOH	92	78

N/D = not determined; RCP = radiochemical purity.

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TABLE 2

Biodistribution of ^99mTc-B2702p1-20 at 180 Minutes After Injection in ApoE^−/− Mice with Left Carotid Artery Ligation

Tracer	Heart	Aorta	Lung	Liver	Spleen	Kidney	Fat	Muscle	Blood	Thyroid	L. carotid	R. carotid
^99mTc-B2702p	1.5 ± 0.1	2.7 ± 0.4	3.9 ± 0.4	10.6 ± 2.6	2.2 ± 0.3	69.0 ± 13.0	1.0 ± 0.2	1.2 ± 0.2	5.7 ± 0.6	9.7 ± 3.0	2.4 ± 0.4	1.8 ± 0.3
^99mTc-B2702p1	0.3 ± 0.1	0.5 ± 0.1	0.8 ± 0.1	3.1 ± 0.6	1.6 ± 0.7	4.6 ± 0.5	0.2 ± 0.0	0.1 ± 0.0	0.9 ± 0.2^†	0.7 ± 0.2	1.3 ± 0.4*	0.5 ± 0.1^†
^99mTc-B2702p1 mismatch	0.7 ± 0.2	1.7 ± 0.7	1.7 ± 0.4	22.1 ± 5.9	0.8 ± 0.1	15.5 ± 4.2	0.5 ± 0.2	0.5 ± 0.1	2.4 ± 0.8	1.1 ± 0.4	1.2 ± 0.6	1.2 ± 0.6
^99mTc-B2702p2	0.3 ± 0.1	0.9 ± 0.6	0.8 ± 0.3	2.7 ± 0.3	1.4 ± 0.4	13.4 ± 1.9	0.3 ± 0.2	0.2 ± 0.1	0.9 ± 0.3^†	1.3 ± 0.1	0.5 ± 0.2^†	0.3 ± 0.1^†
^99mTc-B2702p3	0.4 ± 0.1	0.7 ± 0.2	0.9 ± 0.2	4.2 ± 0.6	0.8 ± 0.2	33.1 ± 0.7	0.3 ± 0.1	0.2 ± 0.1	1.3 ± 0.3^†	0.6 ± 0.1	0.5 ± 0.1^†	0.4 ± 0.1^†
^99mTc-B2702p4	0.3 ± 0.1	0.6 ± 0.3	0.6 ± 0.2	4.7 ± 0.9	0.6 ± 0.2	26.2 ± 5.1	0.2 ± 0.1	0.2 ± 0.1	0.9 ± 0.4^†	0.6 ± 0.2	0.7 ± 0.4^†	0.2 ± 0.1^†
^99mTc-B2702p6	0.3 ± 0.0	0.6 ± 0.0	0.7 ± 0.0	5.9 ± 1.0	0.5 ± 0.0	8.2 ± 0.7	0.2 ± 0.0	0.1 ± 0.0	1.1 ± 0.1^†	1.5 ± 0.3	0.5 ± 0.1^†	0.4 ± 0.2^†
^99mTc-B2702p7	0.4 ± 0.0	0.7 ± 0.1	0.9 ± 0.1	8.2 ± 0.4	1.0 ± 0.3	8.0 ± 1.3	0.2 ± 0.0	0.2 ± 0.0	1.3 ± 0.1^†	1.9 ± 0.7	0.4 ± 0.2^†	0.4 ± 0.2^†
^99mTc-B2702p8	0.3 ± 0.1	0.7 ± 0.1	0.9 ± 0.1	6.9 ± 1.6	0.8 ± 0.2	20.6 ± 10.8	0.2 ± 0.1	0.2 ± 0.1	1.2 ± 0.3^†	1.6 ± 0.6	0.4 ± 0.1^†	0.2 ± 0.1^†
^99mTc-B2702p9	0.1 ± 0.0	0.3 ± 0.0	0.4 ± 0.0	2.1 ± 0.1	0.4 ± 0.1	2.2 ± 0.2	0.1 ± 0.0	0.1 ± 0.0	0.4 ± 0.0^†	0.4 ± 0.0	0.1 ± 0.0^†	0.1 ± 0.0^†
^99mTc-B2702P10	0.5 ± 0.3	0.3 ± 0.0	0.4 ± 0.1	2.8 ± 0.5	0.4 ± 0.1	23.6 ± 3.7	0.1 ± 0.0	0.1 ± 0.0	0.4 ± 0.0^†	0.3 ± 0.0	0.1 ± 0.0^†	0.4 ± 0.2^†
^99mTc-B2702p11	1.8 ± 0.1	2.7 ± 0.1	3.6 ± 0.3	10.8 ± 0.6	2.9 ± 0.1	19.2 ± 0.8	1.5 ± 0.3	0.6 ± 0.1	6.7 ± 0.4	14.2 ± 6.9	2.1 ± 0.3	1.9 ± 0.7
^99mTc-B2702p12	0.4 ± 0.1	2.8 ± 0.9	4.2 ± 1.5	5.5 ± 0.6	3.1 ± 0.2	36.9 ± 11.4	0.8 ± 0.1	0.8 ± 0.4	4.4 ± 1.2	8.8 ± 2.8	2.1 ± 0.9	1.4 ± 0.4
^99mTc-B2702p13	1.6 ± 0.3	2.3 ± 0.3	3.3 ± 0.4	8.0 ± 0.6	2.9 ± 0.4	22.7 ± 7.5	0.8 ± 0.3	0.5 ± 0.1	5.9 ± 0.5	11.0 ± 3.2	1.7 ± 0.3	1.8 ± 0.4
^99mTc-B2702p14	2.5 ± 0.7	3.2 ± 0.6	5.7 ± 1.4	12.7 ± 2.4	3.7 ± 1.0	34.6 ± 1.9	0.8 ± 0.1	0.7 ± 0.2	10.7 ± 2.2	15.9 ± 2.6	2.2 ± 0.2	2.2 ± 0.2
^99mTc-B2702p15	4.5 ± 0.3	5.2 ± 0.1	11.2 ± 0.7	18.1 ± 0.7	6.4 ± 0.2	34.0 ± 4.4	1.6 ± 0.7	1.2 ± 0.1	16.4 ± 0.9	6.3 ± 1.1	3.3 ± 0.7	1.9 ± 0.5
^99mTc-B2702p16	0.3 ± 0.0	0.6 ± 0.1	0.9 ± 0.1	3.4 ± 0.4	1.2 ± 0.1	98.9 ± 18.1	0.4 ± 0.1	0.2 ± 0.0	1.0 ± 0.1^†	1.7 ± 0.2	0.4 ± 0.0^†	0.4 ± 0.2^†
^99mTc-B2702p17	0.4 ± 0.1	0.6 ± 0.2	1.2 ± 0.5	3.5 ± 1.2	1.6 ± 0.7	85.3 ± 25.7	0.3 ± 0.1	0.3 ± 0.2	1.3 ± 0.4^†	2.8 ± 0.4	0.4 ± 0.1^†	0.2 ± 0.1^†
^99mTc-B2702p18	0.5 ± 0.1	2.5 ± 0.4	3.2 ± 0.5	6.3 ± 1.5	2.3 ± 0.7	63.9 ± 17.2	1.2 ± 0.5	0.7 ± 0.0	5.9 ± 1.0	18.8 ± 2.7	2.0 ± 0.2*	1.0 ± 0.1^†
^99mTc-B2702p19	0.5 ± 0.1	0.9 ± 0.3	1.3 ± 0.2	5.5 ± 0.5	1.5 ± 0.1	15.2 ± 6.0	0.2 ± 0.0	0.3 ± 0.0	2.0 ± 0.5^†	1.9 ± 1.5	0.7 ± 0.1^†	1.1 ± 0.5
^99mTc-B2702p20	0.6 ± 0.2	1.4 ± 0.4	1.2 ± 0.3	6.1 ± 1.1	1.0 ± 0.4	11.1 ± 2.0	0.2 ± 0.1	0.2 ± 0.1	2.2 ± 0.6^†	0.5 ± 0.2	0.9 ± 0.1^†	1.2 ± 0.8