Our primary novel finding is the ability to serially image upregulation of iNOS gene expression, in vivo, in vascular inflammation. BLI showed increasing iNOS gene expression over 2 weeks in murine macrophage-rich carotid lesions. We also present a method for inducing vascular inflammation in FVB mice.
Imaging Inflammation in Atherosclerosis by In Vivo BLI
Molecular imaging of vascular inflammation in atherosclerosis has been performed previously in vivo
using single-photon emission computed tomography [12
], fluorescence imaging [13
], positron emission tomography [14
], MRI [15
], and CT [16
]. To our knowledge, BLI of vascular inflammation has not been shown previously. In vivo
BLI has several advantages [6
]: it does not need external light excitation, which offers a high signal to background ratio; its rapid chemical reaction to generate light allows for real-time detection of biological processes; and it can provide a quantitative measure.
The Role of iNOS in Atherosclerosis
iNOS is induced by a variety of inflammatory cytokines and has been shown to be abundantly expressed by macrophages in atherosclerotic lesions [5
]. Several studies have shown that reducing iNOS expression in animal models reduces atherosclerosis [17
]. Based on these and other data, it is thought that upregulation of iNOS in vascular macrophages plays an important role in the progression of atherosclerosis [19
]. In the present study, we have demonstrated serial increase in vascular iNOS expression with the development of a macrophage-rich carotid lesion. Further studies are needed to evaluate in vivo
BLI for monitoring the responses to therapeutic interventions.
Macrophage-Rich Atherosclerotic Lesions in iNOS-luc Mice with Diabetes and High-Fat Diet
There are strain-dependent differences in the vascular response to injury [20
]. The white coat of FVB mice is advantageous for BLI and many existing transgenic luc
mice are on the FVB background. However, this strain typically develops proliferative lesions in response to carotid ligation without significant macrophage infiltration [20
]. Several researchers reported that STZ-induced diabetes can accelerate atherosclerosis in mice mainly through the augmented inflammatory reaction [22
]. The combination of high-fat diet and STZ-induced diabetes successfully generated macrophage-rich carotid-ligation lesions in FVB mice, broadening the available murine models of vascular inflammation.
The model of vascular inflammation we used develops much more rapidly than human atherosclerosis. While this is useful as a preclinical model, it does not fully replicate the complex lesions of human atherosclerotic disease. Our data show an increase in iNOS expression by BLI and histology over 14 days, but further studies of BLI over a longer time course and the response to interventions would be valuable. The effects of age on iNOS expression and this vascular inflammation model also warrant further investigation.
BLI itself has several limitations. It does not directly image iNOS, rather the transcriptional activation of the iNOS promoter. Thus, the BLI signal would be expected to correlate with the number of macrophages and their degree of iNOS activation. It is primarily a 2D projection imaging technique, however, without depth information and with decreased sensitivity to detect deeper structures, which could affect the BLI signal independent of vascular macrophage volume. Newly developed CCD cameras that can reconstruct 3D views or fluorescence molecular tomography systems may be helpful. Furthermore, the limited penetration hinders the use of BLI for human application. However, as shown in this study, it does provide a rapid, high-throughput method to study disease in vivo over time in relevant pre-clinical models.