We report here that monocyte recruitment to plaques can be assessed by microSPECT/CT imaging and thus be visualized in a non-invasive, dynamic, three-dimensional and quantitative fashion. The long half-life of radiotracer isotopes available for SPECT imaging enables repetitive imaging of monocyte biodistribution in the same animal, tracking its migration from circulation to atherosclerotic lesions.
Previous reports have indicated that lesion macrophage content may be gauged by magnetic resonance imaging (MRI) 18, 19
, optical coherence tomography (OCT) 20
or computed tomography (CT) 21
. However, these methods can only describe the total number of resident macrophages or overall metabolic activity, respectively, in a plaque at a given time. As atherogenesis is a chronic and dynamic process, the number of macrophages present in a plaque at a given time is the combined result of monocyte recruitment, differentiation, death, and efflux from the plaque. The same limitation holds true for positron-emission-tomography (PET) studies using fluorodeoxyglucose (FDG), which have shown promise in estimating the overall degree of vascular inflammation 22, 23
. In contrast to these techniques, in vivo
SPECT/CT imaging now enables assessment of monocyte accumulation serially in the same animal, allowing a much more direct study of factors that modulate recruitment.
The method we describe here has several limitations. The resolution of the present approach is lower compared to certain high-resolution modalities such as MRI. Coregistration with CT (as in the current study) and in the future also MRI can reduce this shortcoming by providing greater anatomical detail and hence better spatial localization of the tracer signal.
SPECT/CT imaging of adoptively transferred cells measures the total number of gamma photons in a defined three-dimensional space and assumes an equal number of photons emitted from each cell. The method may underestimate the number of recruited cells as they undergo apoptosis, which may lead to loss of tracer. The known progressive loss of 111
In over time in healthy cells is relatively constant, although some variations may occur in vivo in different disease models. Indium that has lost its association with cells does not significantly influence the local, cell bound activity as our prior studies have shown that free 111
In is rapidly excreted through the kidneys 14
A confounding variable in the interpretation of the results is the possibility that monocytes turn over in atherosclerotic plaques due to continued recruitment and emigration. However, the data of Llodra et al. have shown that while this is true for wild-type mice, there is only minimal emigration of monocytes from plaques in ApoE−/−
. Based on this data we believe that the signal detected in lesions in our study reflects the accumulation of monocytes over the 5-day period from adoptive transfer until the imaging endpoint. We understand that the number of adoptively transferred monocytes (3 × 106
) exceeded the number of circulating monocytes in the recipient animal, which may affect the rate of monocyte trafficking, particularly in the initial hours/days.
Several technical factors were crucial to the development of a robust imaging technique. As monocytes occur in relatively small numbers in the peripheral blood of mice and do not express a known unique surface marker, their isolation represents a challenge. Here we employed a two-step method, recently described and characterized in more detail 14
, that allows high-purity isolation while preserving cell viability and functionality.
Prerequisites for a suitable radioactive tracer used to label monocytes include ready availability, negligible cytotoxic effects, sufficient labeling efficiency, and a long half-life. The radiotracer compound 111In-oxine used here is available in a form ready for coincubation with cells, and is approved by the FDA for labeling autologous leukocytes. Despite extensive testing in vitro and in vivo, we did not find adverse effects on viability or function of monocytes.
In-oxine has a physical half-life (t1/2
= 2.8 days) that is significantly longer than isotopes available for positron-emission-tomography (PET) imaging, such as 64
Cu (12.7 h) or 18
F (110 min) 25
. This translated in our ability to visualize monocyte trafficking by microSPECT/CT for up to 7–10 days, whereas the longest cell-tracking periods possible with state-of-the-art PET imaging have been reported to be 24–36 h 25
except for HSV-tk techniques which require genetic modification of cells. This property has particular importance to the present study, as we were able to use a delayed imaging time-point (5 days) for the readout, avoiding detection of false positive signal derived from circulating monocytes (We had recently determined the half-life of circulating blood monocytes in mice to be 43.5 ± 7.9 h, corresponding to a 95% monocyte clearance from the circulation at the time-point of 5 days after injection 14
The SPECT signal localized predominantly in regions of the ascending aorta, the site of the largest plaques in the majority of animals, while certain small lesions did not emit detectable signal. This observation may result from lesion heterogeneity, i.e. some lesions may be more active than others. This method may therefore be used to identify lesions that are recruiting more monocytes. Alternatively, the total number of cells in smaller lesions is below the SPECT detection threshold. Further improvement of labeling efficiency and improvement in SPECT technology should increase sensitivity in the future.
Having an in vivo imaging system at hand, we could then use it to assess efficiently the effect of modulating factors on monocyte recruitment, avoiding labor-intensive ex vivo procedures.
All three tested statins had an acute effect on monocyte recruitment, rapidly and independently of significant effects on plasma cholesterol levels. To our knowledge this is the first time that such acute statin effects have been reported. Prospective clinical trials have convincingly demonstrated that statins can effectively lower the incidence of cardiovascular events in primary and secondary prevention 26
. An increasing body of evidence suggests that statins cause these clinical benefits in part by anti-inflammatory effects not directly related to the lowering of LDL cholesterol (LDL-C), a hypothesis supported by the present observations. Statin-induced alterations in arterial biology that may not depend on cholesterol lowering include reduction in the expression of factors involved in the recruitment of inflammatory cells, such as MCP-1 17, 27
, ICAM-1 26
, IL-6 and IL-8 28
, TNF-α 17
and NF-κB activity 27
and monocytosis 7
. Several reviews point to uncertainty regarding the in vivo
relevance of such “pleiotropic” effects 26, 29
. Recent analyses of clinical trials however support the concept that an important component of statins’ reduction in recurrent cardiovascular events does not depend on LDL reduction 30, 31
. The relevance of LDL-independent effects of statins also remains unclear because many in vitro studies used statin concentrations too high to have clinical relevance 29
. Most of the studies in mice used dosages between 10–30 mg/kg 32, 33
, which is at least an order of magnitude higher than the clinically prescribed range of 10–80 mg/person/day (0.14 – 1.14 mg/kg for a standard weight of 70 kg). The atorvastatin dosage used here (0.57 mg/kg) corresponds to a 40 mg dose in humans. The current study - using a low dose - showed that atorvastatin causes an immediate and substantial reduction in monocyte recruitment to atherosclerotic plaques. After treating ApoE−/−
mice with only three statin doses, the monocyte recruitment to plaques fell 5-fold in vivo
. This finding has considerable importance, as it highlights the potential of statins to suppress a key step in atherogenesis via an effect independent of LDL-C lowering. Furthermore, it supports the relevance of previous in vitro reports on the effect of statins on mediators of monocyte attachment to and transmigration through the vascular endothelium. Reciprocal experiments treated the donor mice (and therefore exposed the monocytes to atorvastatin but not the vessel wall of the recipients) and showed no significant reduction in monocyte recruitment. This finding suggests that atorvastatin reduces recruitment by acting at the level of the arterial wall rather than on the monocytes themselves. However, the possibility remains that the statin effect on monocytes in vivo may have been lost during monocyte isolation and no longer operate upon adoptive transfer. Therefore we cannot conclude that statins affect exclusively the vascular wall under these conditions.
In summary, we present novel mechanistic insight into monocyte accumulation in atheromata, and the anti-inflammatory action of statins, using a new tool that permits tracking of cells to the vascular wall noninvasively in vivo. Using monocytes in the context of atherogenesis, we exemplify how this technique can be applied to elucidate important biological events. As the key components of the technique are in clinical use, its application to human patients may be within reach.