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Inflammation plays a critical role in atherosclerosis and is associated with upregulation of inducible nitric oxide synthase (iNOS). We studied bioluminescence imaging (BLI) to track iNOS gene expression in a murine model of vascular inflammation.
Macrophage-rich vascular lesions were induced by carotid ligation plus high-fat diet and streptozotocin-induced diabetes in 18 iNOS-luc reporter mice. In vivo iNOS expression was imaged serially by BLI over 14 days, followed by in situ BLI and histology.
BLI signal from ligated carotids increased over 14 days (9.7±4.4×103 vs. 4.4±1.7×103 photons/s/cm2/sr at baseline, p<0.001 vs. baseline, p<0.05 vs. sham controls). Histology confirmed substantial macrophage infiltration, with iNOS and luciferase expression, only in ligated left carotid arteries and not controls.
BLI allows in vivo detection of iNOS expression in murine carotid lesions and may provide a valuable approach for monitoring vascular gene expression and inflammation in small animal models.
Accumulating evidence has established that vascular inflammation plays an important role in atherosclerosis [1, 2]. Inflammation is involved not only in the initiation of atherosclerosis but also its progression and complications. Thus, visualizing active inflammation within the vessel wall may help to identify atherosclerosis at an early stage as well as characterize disease activity and risk.
Nitric oxide (NO) is thought to be a key regulator in the development of atherosclerosis. NO is synthesized from L-arginine by three NO synthase (NOS) isoforms—one inducible form (iNOS) and two constitutive-type isoforms: neuronal (nNOS) and endothelial (eNOS) enzymes. iNOS is barely detectable in the normal vasculature, but it is abundantly expressed in atherosclerosis, primarily by macrophages . Previous studies show that iNOS expression is associated with advanced atherosclerosis and instability of atherosclerotic plaques on histology [4, 5]. Therefore, visualizing iNOS expression in vivo may provide an important approach to detect and follow vascular inflammation and monitor the effects of therapeutic interventions.
In vivo bioluminescence imaging (BLI) is a sensitive technique for small animal molecular imaging. It allows for real-time assessment of gene induction in vivo in models where luciferase expression is controlled by promoter elements of specific genes , including iNOS . In this study, we investigated BLI for monitoring iNOS expression in a murine model of vascular inflammation.
The iNOS promoter-luciferase transgenic mouse (iNOS-luc; a Xenogen product from Caliper Life Sciences ) was used (n=18, 8–12 weeks old). This has a white coat on the FVB/N background and has been shown to have high BLI signal from activated macrophages in vitro and in a model of arthritis in vivo .
To induce a macrophage-rich vascular lesion on the FVB/N background, animals were fed a high-fat diet, containing 40% kcal fat, 1.25% (by weight) cholesterol and 0.5% (by weight) sodium cholate (D12109, Research Diets, Inc. New Brunswick, NJ, USA; Fig. 1) . After 4 weeks of high-fat diet, mice were rendered diabetic by administration of five daily intraperitoneal injections of streptozotocin (STZ), 40 mg/kg in citrate buffers (0.05 mol/L, pH 4.5, Sigma Aldrich) . STZ was given for an additional 3 days if the day 5 serum glucose level was below 200 mg/dL. At day 14, after initiation of STZ injection, the left common carotid artery was ligated (n=14) below the bifurcation with the use of 5–0 silk ligature (Ethicon) under 2% inhaled isoflurane, as previously described . In sham-operated mice (n=4), the suture was passed around the exposed left carotid artery but not tightened. The wound was closed by suture and the animals were recovered on a warming blanket. Animals were followed by serial BLI up to 14 days after ligation. All procedures were approved by the Administrative Panel on Laboratory Animal Care at Stanford University.
BLI was performed serially at days 0 (pre), 3, 5, 7, 10, and 14, as previously described [6, 11]. Briefly, animals were anesthetized (2% inhaled isoflurane) and luciferin (10 μg/g) was administered intraperitoneally. Seven to ten minutes later, anesthetized animals were imaged for 5 min using an IVIS200 charge-coupled device imaging system (Xenogen Corp. part of Caliper Life Sciences, USA). At day 14, in situ BLI was also performed: mice were injected with an additional dose of luciferin (10 μg/g, intraperitoneally), then euthanized, and both carotid arteries rapidly exposed and imaged for 5 min beginning at 7–10 min after luciferin injection. Imaging data were analyzed by LivingImage software (Xenogen Corporation, USA) and IgorPro software (WaveMetrics, USA). For quantitative analysis, a region of interest over the left common carotid artery was outlined and the total number of photons in the region was measured, expressed as photons(p)/s/cm2/sr.
After in situ BLI on day 14, animals were perfusion fixed with 10% phosphate-buffered formalin at physiological pressure. Both right and left common carotid arteries were removed with the heart and further fixed in 10% formalin for 24 h. The left common carotid artery from 1 to 4 mm below the ligation was removed, cut into three portions (1-mm intervals), and embedded into paraffin for serial sectioning (10 μm). The right common carotid artery, as an internal control, was processed similarly. Macrophages were immunostained by rat anti-mouse Mac-3 monoclonal antibody (#550292, BD Pharmingen). iNOS expression was immunostained by rabbit polyclonal antibody against iNOS (sc-651, Santa Cruz Biotechnology, Inc. CA, USA). Luciferase was immunostained by rabbit polyclonal antibody against luciferase (sc-32896, Santa Cruz Biotechnology, Inc. CA, USA).
Data are expressed as mean±SD. One-way repeated measures ANOVA with Fisher's post-hoc test was used for analyzing the serial BLI data of iNOS expression. Two-way factorial ANOVA with Fisher's post-hoc test was used to compare BLI data between ligated and sham-operated mice. P<0.05 was considered to be statistically significant.
The ligated left common carotid arteries demonstrated macrophage-rich lesions proximal to the suture (Fig. 2), which were not evident in the sham or contralateral (non-ligated) controls. Immunohistochemistry in ligated arteries showed substantial macrophage infiltration of both the neointima and adjacent to the adventitia with increased iNOS expression in the same regions, in contrast to only a small number of adventitial macrophages and no significant iNOS expression in the sham controls. Immunohistochemistry of luciferase (Fig. 3) confirmed a similar pattern of iNOS-luc gene expression in the ligated carotid but not in control.
The BLI signal from the neck of ligated mice localized to the left common carotid artery (Fig. 4a), which was further confirmed by in situ BLI at day 14 (Fig. 4b). Sham-operated mice had only transient BLI signal noted over the midline suture (Fig. 4c). Quantitative BLI analysis (Fig. 5) showed increasing BLI signal from the ligated mice over the 14 days compared to pre-ligation (pre: 4.4±1.7×103 p/s/cm2/sr; day 5: 7.6±2.8×103 p/s/cm2/sr, p=0.0007; day 14: 9.7±4.4×103 p/s/cm2/sr, p<0.0001). Sham-operated mice (Fig. 5b), in contrast, showed a transient increase in BLI signal peaking at day 5 (5.8±1.5×103 p/s/cm2/sr, p=0.02 vs. pre), then returning to baseline (day 7: 4.6±0.2×103 p/s/cm2/sr, p=0.4 vs. pre). Comparing ligated to sham-operated mice, BLI signal was significantly higher in ligated arteries after day 7 (p<0.05, Fig. 5).
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.
Molecular imaging of vascular inflammation in atherosclerosis has been performed previously in vivo using single-photon emission computed tomography , fluorescence imaging , positron emission tomography , MRI , and CT . To our knowledge, BLI of vascular inflammation has not been shown previously. In vivo BLI has several advantages : 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.
iNOS is induced by a variety of inflammatory cytokines and has been shown to be abundantly expressed by macrophages in atherosclerotic lesions . Several studies have shown that reducing iNOS expression in animal models reduces atherosclerosis [17, 18]. 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 . 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.
There are strain-dependent differences in the vascular response to injury . 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, 21]. Several researchers reported that STZ-induced diabetes can accelerate atherosclerosis in mice mainly through the augmented inflammatory reaction [22, 23]. 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.
BLI can image increased iNOS gene expression in vivo in murine vascular inflammation. BLI has the potential to noninvasively monitor gene expression and inflammation in experimental atherosclerosis, which may aid in the development and evaluation of novel therapies.
We thank Dr. Timothy Doyle for his technical assistance with in vivo bioluminescence imaging. This work was supported by a grant from the National Institutes of Health, R01-HL078678.
This work is significant for demonstrating serial in vivo imaging of iNOS expression in vascular inflammation.
Disclosures. Dr. Terashima has received honoraria from Philips Healthcare. Dr. McConnell's laboratory receives research support from GE Healthcare and he is on a scientific advisory board for Kowa, Inc. Dr. Quertermous is a founder, stockholder, and on the scientific advisory board for VIA Pharmaceuticals. Dr. Contag is a founder and consultant for Xenogen Corporation (now Caliper Life Sciences). The other authors have no potential conflicts of interest.