18F-4V for PET–CT Imaging of VCAM-1 Expression in Atherosclerosis
Matthias Nahrendorf, MD, PhD*, , ,
Edmund Keliher, PhD*,,
Peter Panizzi, PhD,
Hanwen Zhang, PhD,
Sheena Hembrador, BS,
Jose-Luiz Figueiredo, MD*,,
Elena Aikawa, MD,
Kimberly Kelly, PhD,
Peter Libby, MD, ,
Ralph Weissleder, MD, PhD*,,,*
* Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts
Donald W. Reynolds Cardiovascular Clinical Research Center on Atherosclerosis at Harvard Medical School, Boston, Massachusetts
Cardiovascular Division, Department of Medicine, Brigham & Women's Hospital, Boston, Massachusetts
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Abstract
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Objectives: The aim of this study was to iteratively develop and validate an 18F-labeled small vascular cell adhesion molecule (VCAM)-1 affinity ligand and demonstrate the feasibility of imaging VCAM-1 expression by positron emission tomography–computed tomography (PET-CT) in murine atherosclerotic arteries.
Background: Hybrid PET-CT imaging allows simultaneous assessment of atherosclerotic lesion morphology (CT) and may facilitate early risk assessment in individual patients. The early induction, confinement of expression to atherosclerotic lesions, and accessible position in proximity to the blood pool render the adhesion molecule VCAM-1 an attractive imaging biomarker for inflamed atheroma prone to complication.
Methods: A cyclic, a linear, and an oligomer affinity peptide, internalized into endothelial cells by VCAM-1–mediated binding, were initially derivatized with DOTA to determine their binding profiles and pharmacokinetics. The lead compound was then 18F-labeled and tested in atherosclerotic apoE–/– mice receiving a high-cholesterol diet as well as wild type murine models of myocardial infarction and heart transplant rejection.
Results: The tetrameric peptide had the highest affinity and specificity for VCAM-1 (97% inhibition with soluble VCAM-1 in vitro). In vivo PET-CT imaging using 18F-4V showed 0.31 ± 0.02 SUV in murine atheroma (ex vivo %IDGT 5.9 ± 1.5). 18F-4V uptake colocalized with atherosclerotic plaques on Oil Red O staining and correlated to mRNA levels of VCAM-1 measured by quantitative reverse transcription polymerase chain reaction (R = 0.79, p = 0.03). Atherosclerotic mice receiving an atorvastatin-enriched diet had significantly lower lesional uptake (p < 0.05). Furthermore, 18F-4V imaging in myocardial ischemia after coronary ligation and in transplanted cardiac allografts undergoing rejection showed high in vivo PET signal in inflamed myocardium and good correlation with ex vivo measurement of VCAM-1 mRNA by quantitative polymerase chain reaction.
Conclusions: 18F-4V allows noninvasive PET-CT imaging of VCAM-1 in inflammatory atherosclerosis, has the dynamic range to quantify treatment effects, and correlates with inflammatory gene expression.
Key Words: atherosclerosis • inflammation • molecular imaging • PET-CT • VCAM-1
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Abbreviations and Acronyms
| | 18F-4V = 18F-labeled tetrameric peptide-PET imaging reporter targeted to VCAM-1 | | 18FDG = [18F]-fluorodeoxyglucose | | ApoE = apolipoprotein E | | DOTA = 1,4,7,10-tetraazadodecane-1,4,7,10-tetraacetic acid, chelator for 111-Indium labeling | | MCP = monomeric cyclic peptide (DOTA-labeled) | | MHEC = murine heart endothelial cells | | MLP = monomeric linear peptide (DOTA-labeled) | | MI = myocardial infarction | | mRNA = messenger ribonucleic acid | | %IDGT = percent injected dose/gram tissue | | PET-CT = positron emission tomography-computed tomography | | RT-PCR = reverse transcription polymerase chain reaction | | TLP = tetrameric linear peptide (DOTA-labeled) | | VCAM = vascular cell adhesion molecule |
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A reliable noninvasive diagnostic strategy for detecting inflamed arterial lesions at risk for complications could help target and evaluate therapies to prevent myocardial infarction (MI) and stroke. Current clinical imaging technologies largely provide structural information (1); however, the anatomical severity of stenosis does not sufficiently gauge the risk of vascular events (2). Molecular imaging approaches now in development aim to interrogate biological processes rather than morphology (1,3,4).
Vascular cell adhesion molecule (VCAM)-1 plays a cardinal role in atherosclerotic plaque progression (5–7). Activated endothelial cells that line the tissue-blood interface express VCAM-1, as can lesional macrophages and smooth muscle cells (5–7). VCAM-1 mediates inflammatory cell adhesion through interaction with the integrin very late antigen-4 (8). The early induction, confinement of expression to atherosclerotic lesions, and accessible position in proximity to the blood pool render VCAM-1 an attractive imaging biomarker.
We (9,10) and others (11,12) have imaged VCAM-1 as a proof-of-principle in inflammatory disease, for instance with targeted nanoparticles for magnetic resonance imaging (MRI). Although providing early efficacy data, these agents face practical regulatory hurdles that prevent rapid clinical development. Despite the specific advantages of positron emission tomography (PET), the increasing clinical availability of PET-computed tomography (CT) scanners, and the unmet need for noninvasive identification of high-risk vascular lesions, relatively few targeted PET agents exist for plaque imaging (13,14). [18F]-2-fluorodeoxyglucose (18FDG) can accumulate in atherosclerotic lesions (15–17) and is clinically approved for cancer imaging. 18FDG uptake presumably indicates glucose transport, and uptake associates with macrophage (18) and neovessel content (19). Studies in patients undergoing endarterectomy (17) showed increased 18FDG signal in macrophage-rich carotid arteries. However, there remains a need for development of agents that selectively target inflammation in plaques and that have lower background uptake in metabolically highly active myocardial tissue than 18FDG to facilitate coronary imaging.
Here we describe the design, synthesis, evaluation, and use of a new PET imaging agent with optimized pharmacokinetics and specificity for VCAM-1. The overall design of this peptide-based agent hinged on: 1) preference of PET-CT as a hybrid clinical imaging modality with high-sensitivity (PET) combined with detailed anatomical information (CT); 2) choice of 18F as a clinical PET tracer with a short half-life; 3) harnessing powerful signal amplification strategies (multivalency of affinity ligand and VCAM-1–mediated cell internalization); and 4) choice of a probe design that would ultimately allow for rapid clinical translation.
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Methods
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Agent synthesis.
A number of VCAM-1 specific peptide sequences have been identified by phage display technology (9,10) containing linear and cyclic heptapeptides (Table 1). To facilitate comparative testing of agents in the current work, we first derivatized 3 lead peptides with the chelator 1,4,7,10-tetraazadodecane-1,4,7,10-tetraacetic acid (DOTA) and labeled them with 111Indium. Peptides were synthesized with standard N -9-fluorenylmethoxycarbonyl chemistry, followed by high-performance liquid chromatographic analysis, which demonstrated >98% purity. The labeling yields of 111In-DOTA derivatives were >99% at specific activities of 30.8 GBq/µmol. On the basis of initial comparative results, we then redesigned the best peptide (sequence VHPKQHR, linker GGSYKKK, tetramer) and labeled it with 18Fluorine with a benzaldehyde method (20). The synthesis of the lead compound, named 18F-4V, was automated with a PETsynthRN synthesizer (Nebeling GmbH, Drolshagen, Germany) followed by high-performance liquid chromatographic purification. We also synthesized a fluorescent version of 18F-4V by conjugating Cy5 maleimide to enable fluorescence microscopy detection of the probe in histological sections.
Competition assays.
We evaluated the affinity of peptides in competition assays with murine VCAM-1 immobilized on agarose beads. Tetrameric linear peptide (TLP)-DOTA-111In (37 GBq/µmol, 0.5 nmol/l) was added to VCAM-1/agarose beads, and competed off with increasing concentrations of unlabeled monomeric cyclic peptide (MCP)-DOTA, monomeric linear peptide (MLP)-DOTA, and TLP-DOTA, respectively. After washing, the amount of labeled TLP-DOTA-111In bound to beads was quantitated via gamma well counter. Next, we assessed binding of candidate peptides to VCAM-1–expressing endothelial cells. The 111In-labeled peptides were added to murine heart endothelial cells (MHEC) (21) and incubated at 37°C for 1 h. Cells were harvested from the plates, and the radioactivity was measured via gamma well counter. To evaluate specificity for VCAM-1, 18F-4V (0.20 µg) was pre-incubated with 5x murine VCAM-1 or saline and then added to MHEC.
Mouse models.
Apolipoprotein E (ApoE)–/– mice had an average age of 45 weeks and were on a high-cholesterol diet (Harlan Teklad, Madison, Wisconsin), which produces reliable VCAM-1 expression in atherosclerotic plaques located in the aortic root (10). To test imaging in the setting of therapy, we treated a cohort of apoE–/– mice with atorvastatin (enriched in diet, 0.01% wt/wt) (10). Two additional disease entities known to increase VCAM-1 expression were imaged: MI was induced by coronary ligation (22), and heterotopic allograft heart transplantation from BALB/C into C57/B6 mice was performed (23). Mice were anesthetized for all procedures (isoflurane 2% to 3% v/v, Baxter). The institutional subcommittee on research animal care approved all animal studies.
Biodistribution Studies
The blood half-life of candidate probes was determined with serial retro-orbital bleeds after injection of 150 µCi of a given agent into the tail vein of 6 wild-type mice. After sacrifice (4 h), mice were perfused with 10 ml of saline. Organs were harvested, and their activity was recorded with a gamma counter (1480 Wizard 3-inch, PerkinElmer, Waltham, Massachusetts). Biodistribution data were corrected for decay and residual activity at the injection site. Oil Red O staining depicted the distribution of plaques in apoE–/– aortas, which were subsequently analyzed by digital autoradiography. To evaluate the in vivo specificity of 18F-4V, cohorts of mice were pre-injected with an antibody targeted to VCAM-1 (BD Pharmingen, San Diego, California), followed by 18F-4V 60 min later.
Fluorescence and Immunohistology
One hour after injection of Cy5-labeled 125-µg peptide, aortas were harvested for fluorescence microscopy and immunohistochemical detection of VCAM-1 (CD106), endothelial cells (CD31), macrophages (MAC-3) (all BD Pharmingen), and smooth muscle cells (-actin, Lab Vision, Fremont, California).
PET-CT imaging.
PET imaging was initiated 1 h after injection of 18F-4V (325 ± 167 µCi in 200 ± 65 µl) in conjunction with high-resolution vascular CT (Inveon, Siemens, Munich, Germany). The PET data were reconstructed with ordered subsets expectation maximization and filtered back projection algorithms (24), with a spatial resolution approaching approximately 1 mm. For quantitation of PET signal, regions of interest were placed in the aortic root on the basis of anatomical CT imaging. The CT X-ray source was used with a power of 80 kVp and 500 µA, an exposure time of 370 to 400 ms, and an isotropic resolution of 90 µm. During CT acquisition, Isovue-370 (Bracco Diagnostics, Princeton, New Jersey) was infused intravenously.
Quantitative reverse transcription polymerase chain reaction.
To validate in vivo PET data, we correlated 18F-4V uptake to expression of VCAM-1 and CD68 quantified by multiplex quantitative polymerase chain reaction (PCR) (TaqMan, Applied Biosystems, Foster City, California) with glyceraldehyde-3-phosphate dehydrogenase as an endogenous control.
Statistics.
Results are expressed as mean ± SEM. Unpaired data were compared with the unpaired 2-sided t test, and paired data were compared with the paired 2-sided t test. The significance level in all tests was 0.05. Due to space limitations of the format, the Methods section has been abbreviated.
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Results
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In vitro and in vivo screening of candidate peptides.
To determine the affinity of candidate compound, we measured displacement of radioactively tagged TLP bound to purified VCAM-1 by cold MLP, MCP, or TLP. Sigmoidal concentration-dependence curves were observed with an R2 of 0.984 (TLP), 0.9880 (MLP), and 0.9934 (MCP) (Fig. 1A). The tetrameric, linear peptide formulation TLP had a half-maximal inhibitory concentration for VCAM-1 of 86.6 nmol/l, 232- and 349-fold better than MCP or MLP, respectively.
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Figure 1 Screening of Affinity Peptides
(A) Affinity curves for ligands assessed by competition after binding to immobilized vascular cell adhesion molecule (VCAM)-1. Tetrameric linear peptide (TLP) has the highest affinity: counts/min (CPM) and mean ± 95% confidence interval. (B) Cell assay after incubation of murine heart endothelial cells (MHEC) show highest uptake for TLP. (C) Apolipoprotein E (ApoE)–/– mice received injection with 111In-labeled TLP, monomeric cyclic peptide (MCP), and monomeric linear peptide (MLP). Highest autoradiography (Autorad) signal was observed in the root of excised aortas from mice injected with TLP. (D) Percent injected dose/gram tissue (%IDGT) in apoE–/– for TLP, MCP, and MLP. Highest %IDGT was observed for TLP. Data are displayed as mean ± SEM, *p < 0.05.
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We then screened candidates against MHEC, which constitutively express high levels of VCAM-1 (21), and observed a significantly higher accumulation of TLP than MLP or MCP (Fig 1B). Finally, candidate peptides injected into apoE–/– mice facilitated evaluation of uptake into atheroma. TLP showed the highest percent injected dose/gram tissue (%IDGT) in excised aortas 60 min after injection (4.8-fold higher than MLP and 2.4-fold higher than MCP), corroborated by highest signal on autoradiography exposure (Figs. 1C and 1D).
Specificity, biodistribution, and half-life of 18F-4V.
We next proceeded to synthesize the PET tracer 18F-4V by labeling the lead peptide TLP with 18F via 4-[18F]-fluorobenzaldehyde (Fig. 2). Cell uptake experiments involving inhibition with soluble VCAM-1 resulted in a 97% activity decrease, demonstrating a specificity of 18F-4V (Fig. 3A).
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Figure 2 Structure and Purity of 18F-4V
(A) Three-dimensional model of 18F-labeled tetrameric peptide-postitron emission tomography imaging reporter targeted to vascular cell adhesion molecule-1 (18F-4V). The tracer 18F is located on the top, and the 4 branching affinity peptides point downward. (B) Original high-performance liquid chromatographic trace documents purity of the 18F-4V synthesis product.
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Figure 3 Validation of 18F-4V
(A) Pre-incubation of 18F-4V with soluble VCAM-1 reduces uptake into MHEC. (B) Uptake of 18F-4V by scintillation counting. The highest signal was found in aortas of apoE-deficient mice. Statin treatment significantly reduced uptake. Pre-injection of a monoclonal VCAM-1–targeted antibody (MAb) inhibited uptake of 18F-4V. Mean ± SEM, *p < 0.05. (C) Autoradiography corroborates the highest uptake of 18F-4V in apoE-deficient mice, with little uptake in wild-type aortas. Oil Red O staining shows peak uptake in plaques located in the aortic root, arch, and at the renal artery branch. Abbreviations as in Figures 1 and 2.
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We injected 18F-4V into 6 wild-type mice to established the blood half-life and biodistribution. 18F-4V had a blood half-life of 16 ± 0.6 min (R2 of fit = 0.96). The biodistribution at 4 h transpired as follows: (%IDGT): kidney, 13.2 ± 2.8; liver, 3.7 ± 1.1; lymph node, 3.7 ± 0.3; spleen, 2.1 ± 0.6; lung, 2.2 ± 0.8; small intestine 1.9 ± 0.4; fat, 1.7 ± 0.1; skin, 1.7 ± 0.6; stomach, 1.7 ± 1.1; esophagus, 1.5 ± 0.5; blood, 1.5 ± 0.4; large intestine, 1.4 ± 0.5; aorta, 1.3 ± 0.4; bone, 1.3 ± 0.4; thymus 0.9 ± 0.3; skeletal muscle, 0.7 ± 0.3; heart, 0.6 ± 0.2; and feces, 0.2 ± 0.1.
18F-4V accumulates in atherosclerotic plaques.
We assessed uptake of 18F-4V into atherosclerotic plaques in excised aortas of wild-type mice, apoE–/– mice, and apoE–/– mice treated with atorvastatin. Uptake in the aortic root was 312% higher in apoE–/– mice (p < 0.05) (Fig. 3B), when compared with wild type. Autoradiography and en face Oil Red O staining confirmed that activity concentrated in atherosclerotic plaques (Fig. 3C). Treatment with atorvastatin significantly reduced uptake of 18F-4V (Fig. 3B).
To assess the selectivity of in vivo uptake of 18F-4V for VCAM-1, we pre-injected 250 µg of monoclonal VCAM-1 antibody into apoE–/– mice. This procedure reduced the uptake of the agent to levels seen in wild-type mice (Fig. 3B).
The cellular and microscopic distribution of the agent was assessed with a fluorescently labeled version of the tetrameric peptide and showed good correlation with immunoreactive VCAM-1 expression (Fig. 4). The probe distributed mainly to endothelial and subendothelial layers and colocalized primarily with endothelial cells.
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Figure 4 Histologic Probe Distribution
A fluorescent version of 18F-4V (Cy5-4V) was used to explore microscopic and cellular agent distribution. In fluorescence micropscopy, the endothelial and subendothelial layers of an atherosclerotic plaque in the aortic root show strong uptake, whereas autofluorescence in the FITC channel is negligible. On adjacent sections, VCAM-1 and endothelial staining (CD31) colocalize with the agent, with some uptake in macrophages (MAC-3) and smooth muscle cells (-actin). Magnification 400x.
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In vivo PET-CT imaging of 18F-4V detects VCAM-1 expression in atherosclerotic plaques.
Dynamic PET imaging identified 60 to 120 min after injection as the optimal time period for acquisition (Fig. 5). At that time, we found a strong focal PET signal in the aortic root of apoE–/– mice (Fig. 6). Hybrid imaging facilitated unambiguous allocation of the PET signal to the vascular bed of interest identified by contrast-enhanced vascular CT. Absolute quantification of PET signal (standard uptake value) showed significantly higher values in the root of apoE–/– compared with atorvastatin-treated apoE–/– or wild-type mice (Fig. 6).
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Figure 5 Dynamic PET-CT Imaging
Dynamic positron emission tomography (PET) imaging identified 60 to 120 min after injection as a suitable time window, with low blood signal and high activity in the target. The blood activity was measured in the left ventricular blood pool. Imaging signal in the blood pool and the aortic region is plotted (A) and shown over time in short- (B) and long-axis (C) PET–computed tomography (CT) acquisitions. Initially, acquisition times were shorter due to high count rates, whereas progressing decay of 18F necessitated longer acquisition times in later acquisitions.
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Figure 6 PET-CT in ApoE–/– and Statin-Treated Mice
PET-CT imaging shows uptake of 18F-4V in the aortic root (arrows) and arch of atherosclerotic mice. Uptake is lower in statin-treated and in wild-type mice. (A, C, E) Short-axis views. (B, D, F) Long-axis views. (G, H, I) Three-dimensional maximum intensity projection. Bone is shown in white, vasculature in blue, and 18F-4V PET signal in red. The PET signal occurs in the carotid arteries, and background signal in the liver, in addition to the strong uptake of 18F-4V PET observed in the root and arch (arrow). K = quantification of PET signal as the standard uptake value (SUV). Mean ± SEM, *p < 0.05. Abbreviations as in Figures 1, 2, and 5.
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Uptake of 18F-4V closely correlates with inflammatory gene expression.
After imaging, we determined the ex vivo activity of aortic sections and assessed gene expression by reverse transcription (RT)-PCR (Fig. 7). 18F-4V–derived activity correlated with VCAM-1 mRNA levels (R = 0.79, p = 0.03) (Fig. 7). We also explored gene expression of CD68, a macrophage biomarker of inflammatory atherosclerosis (R2 = 0.50, p < 0.05).
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Figure 7 Correlation of 18F-4V Uptake With VCAM-1 Gene Expression
Correlation of 18F-4V uptake to VCAM-1 mRNA levels in imaged vascular segments. After measuring uptake by scintillation counting, gene expression was determined by quantitative RT PCR. When compared with VCAM-1 expression in the root of high-cholesterol–fed apoE-deficient mice, atorvastatin significantly reduced expression, which was very low in wild-type mice. VCAM-1 mRNA levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene. Data are displayed as mean ± SEM, *p < 0.05. The lower panel depicts a close correlation of VCAM-1 mRNA level with uptake of 18F-4V. AU = arbitrary units; other abbreviations as in Figures 1 and 2.
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VCAM-1 imaging in other cardiovascular disorders.
Given the aforementioned results, we reasoned that 18F-4V PET imaging could apply to a variety of other cardiovascular conditions (e.g., ischemic myocardial injury or transplant rejection), situations that might involve widespread VCAM-1–mediated monocyte recruitment. We assayed VCAM-1 transcript levels in mice 5 days after MI and on day 7 after heart transplantation. The VCAM-1 mRNA levels increased 20- and 3-fold, respectively. As in atherosclerosis, inflamed myocardium had considerable uptake of 18F-4V (Fig. 8). The specificity of this uptake was investigated in vivo with blocking of uptake by pre-injection with a VCAM-1 targeted antibody, which reduced the infarct activity/background ratio from 3.1 to 1.4 and, in the transplant model, the graft/background ratio from 2.4 to 1.4.
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Figure 8 18F-4V Imaging in MI and Transplant Rejection
(A) PET-CT shows strong signal in the infarcted left ventricular wall. (B) Infarcted myocardium shows delayed CT hyperenhancement after iodine (arrows). (C) Autoradiography of myocardial ring. (D) %IDGT in the infarct. (E) VCAM-1 mRNA in infarct tissue. (F, G) PET-CT of a heart transplanted heterotopically into the abdominal cavity. The rejected allograft (arrowheads) shows high uptake of 18F-4V. (H, I) Autoradiography of graft and orthotopic recipient heart. (K) Uptake of 18F-4V in rejected allografts. (L) VCAM-1 mRNA levels in control heart tissue and rejected cardiac allografts. *p < 0.05. MI = myocardial infarction; other abbreviations as in Figures 1, 2, 5, and 7.
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Discussion
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The central role of VCAM-1 in the evolution of inflammatory vascular lesions and its exposed accessible position on the endothelial surface render this adhesion molecule an attractive imaging target for atherosclerosis, MI, and transplant rejection. Here we show that PET-CT can image VCAM-1 and describe the design, synthesis, and validation of the novel PET imaging agent 18F-4V. The technique detects VCAM-1 expression in murine aortas, vessels with a diameter considerably smaller than epicardial human coronary arteries. We also show that targeting VCAM-1 is useful for imaging of other cardiovascular diseases.
The initial design of 3 candidate probes derived from peptides identified by phage display (9,10). Although all of these peptides did target VCAM-1, the modified imaging probes differed in their affinity, molecular weight, and pharmacokinetics. TLP, the lead peptide with the highest affinity and most favorable pharmacokinetics, had an arborizing, tetrameric design. The multivalency of TLP resulted in superior performance, a phenomenon previously observed for targeted nanoparticles (25). Consequently, we derivatized TLP with the clinical PET tracer 18F and investigated 18F-4V in a number of in vitro and in vivo experiments. In vitro, pre-incubation of the agent with soluble VCAM-1 almost completely blocked cellular uptake. In vivo, pre-injection of a VCAM-1–specific antibody blocked 18F-4V uptake, and 18F-4V signal correlated closely with VCAM-1 mRNA levels in respective vascular territories (R = 0.79). Areas of highest activity colocalized with Oil Red O–stained atherosclerotic plaques on excised aortas. Statin treatment, known to reduce VCAM-1 expression (26), diminished the ex vivo and in vivo 18F-4V signal. The biodistribution of 18F-4V proved favorable, with low background uptake in undiseased vessel walls and a short blood half-life, which will allow for rapid injection-imaging sequences.
VCAM-1 expression contributes to the pathophysiology of a variety of other cardiovascular conditions—for instance to inflammation after ischemic injury (11,22,27). Myocardial infarction triggers a profound influx of neutrophils and monocytes on days 1 to 6 after ischemia, and the quantity as well as quality of the myeloid cell influx determine the degree of ensuing heart failure and therefore prognosis (27–29). As an integral part of the recruiting mechanism for monocytes, VCAM-1 expression could gauge the degree of inflammation after MI. We found a substantial increase of VCAM-1 mRNA levels in mice on day 5 after coronary ligation, consistent with 18F-4V accumulation.
VCAM-1 also rises during cardiac transplant rejection and promotes monocyte recruitment into the graft (30,31). Mononuclear phagocytes constitute up to 60% of the inflammatory cell population during parenchymal rejection (32). Patients after heart transplantation would greatly benefit from noninvasive assessment of rejection, because repetitive endomyocardial biopsies, the current reference standard, involve invasion and carry a risk of complication. VCAM-1 mRNA levels increased in heterotopically transplanted cardiac allografts, and the 18F-4V signal indeed rose in these rejecting allografts.
Compared with previous efforts to visualize VCAM-1 by MRI or ultrasound (9,10,12,33), 18F-4V imaging provides a number of potential advantages. Positron emission tomography affords absolute signal quantification and will allow robust and noninvasive measurement of VCAM-1 expression, critical for assessment of individual patients as well as patient cohorts in clinical trials. Not only do high affinity and specificity for a target essential to lesion biology support the translatability of 18F-4V, so too does its employment of a clinically established PET tracer and its clinically useful probe design. The inherent sensitivity of PET allows detection of targets at concentrations that are several orders of magnitude lower than are seen with other modalities, for instance MRI. This feature might have particular importance when targets are as small as atherosclerotic plaques.
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Acknowledgments
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The authors acknowledge the help of Peter Waterman, BS, Gregory Wojtkiewicz, MS, Brett Martinelli, BS, Yoshiko Iwamoto, BS, and Timur Shtatland, PHD.
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Footnotes
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This work was funded in part by the D.W. Reynolds Foundation (to Drs. Libby and Weissleder), UO1-HL080731 (to Dr. Weissleder), R01-HL078641 (to Dr. Weissleder), and R24-CA92782 (to Dr. Weissleder). Drs. Keliher, Panizzi, and Zhang contributed equally to this article.
* Reprint requests and correspondence: Dr. Ralph Weissleder, Massachusetts General Hospital, Center for Systems Biology, CPZN-5206, 185 Cambridge Street, Boston, Massachusetts 02124 (Email: rweissleder{at}mgh.harvard.edu).
Manuscript received March 2, 2009;
revised manuscript received March 31, 2009,
accepted April 23, 2009.
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