Visualization of Synthetic Vascular Smooth Muscle Cells in Atherosclerotic Carotid Rat Arteries by F-18 FDG PET

Synthetic vascular smooth muscle cells (VSMCs) play important roles in atherosclerosis, in-stent restenosis, and transplant vasculopathy. We investigated the synthetic activity of VSMCs in the atherosclerotic carotid artery using 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET). Atherosclerosis was induced in rats by partial ligation of the right carotid artery coupled with an atherogenic diet and vitamin D injections (2 consecutive days, 600,000 IU/day). One month later, rats were imaged by F-18 FDG PET. The atherosclerotic right carotid arteries showed prominent luminal narrowing with neointimal hyperplasia. The regions with neointimal hyperplasia were composed of α-smooth muscle actin-positive cells with decreased expression of smooth muscle myosin heavy chain. Surrogate markers of synthetic VSMCs such as collagen type III, cyclophilin A, and matrix metallopeptidase-9 were increased in neointima region. However, neither macrophages nor neutrophils were observed in regions with neointimal hyperplasia. F-18 FDG PET imaging and autoradiography showed elevated FDG uptake into the atherosclerotic carotid artery. The inner vessel layer showed higher tracer uptake than the outer layer. Consistently, the expression of glucose transporter 1 was highly increased in neointima. The present results indicate that F-18 FDG PET may be a useful tool for evaluating synthetic activities of VSMCs in vascular remodeling disorders.

population of VSMCs. 18 F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is an established noninvasive image modality for measuring vascular glucose consumption 4 . In the present study, therefore, we investigated synthetic VSMC activities using F-18 FDG PET in an atherosclerotic rat model of partial carotid artery ligation.
Normal artery was composed of smooth muscle myosin heavy chain (SM-MHC) and α-smooth muscle actin (α-SMA)-positive VSMCs in media (Fig. 2). The neointima in atherosclerotic right carotid artery exhibited many α-SMA-positive VSMCs, whereas SM-MHC-positive VSMCs, CD68-positive macrophages or myeloperoxidase (MPO)-positive neutrophils were scantly observed (Fig. 3). The neointimas exhibited significant collagen type III deposition (Fig. 4), which has been shown to be mainly excreted by synthetic VSMCs 11 . Furthermore, the thickened neointima showed increased expression of cyclophilin A and matrix metallopeptidase-9 (MMP-9) (Fig. 4), both of which are established hallmarks of synthetic VSMC activity 12, 13 . F-18 FDG PET. Increased FDG uptake was observed along the atherosclerotic (right) carotid artery ( Fig. 5A and B). The corresponding maximum SUV was 3.28 ± 0.43 (mean ± SD). As shown in the transverse view in Fig. 5B-a, the inner circular layer of the right carotid artery showed higher tracer uptake than the outer layer. In the normal control group, no FDG uptake was apparent in either the right or left carotid artery ( Fig. 5A and B). The corresponding maximum SUV was 0.86 ± 0.1 (mean ± SD). Atherosclerotic carotid artery showed significantly higher maximum SUV than the normal control group (p = 0.029, Fig. 5C).
Autoradiography and glucose transporter 1 (GLUT1). Consistent with the in vivo PET imaging, autoradiography showed much higher radioactivity in the right carotid artery than in the left carotid artery (Fig. 6A). Furthermore, higher radioactivity was observed in the inner layer compared to the outer layer. Western blotting showed increased GLUT1 expression in atherosclerotic right carotid artery (Fig. 6B). Immunohistochemistry further demonstrated that GLUT1 expression was increased by synthetic VSMCs in neointimal region of atherosclerotic right carotid artery (Fig. 6C).

Discussion
In the atherosclerotic rat model used in the present study, the neointima became hypertrophic and exhibited a large VSMC population. However, few inflammatory cells such as macrophages or neutrophils were observed. Our findings are similar to those previously found in a high fat diet-induced atherosclerotic rat model 10 . Using F-18 FDG PET, we found that VSMCs from the neointima exhibited the synthetic phenotype rather than the contractile phenotype.
When blood vessels are damaged, VSMCs can switch from the contractile to the synthetic phenotype 2, 3 . During the process of neointimal hyperplasia, SM-MHC which is a well-known marker of contractile VSMC is decreased in neointima whereas α-SMA expression is preserved [14][15][16] . Our results are consistent with these previous studies. In the present study, we further found that surrogate markers of synthetic VSMCs such as collagen type III, cyclophilin A, and MMP-9 were increased in the neointima. Therefore, synthetic VSMCs appear to constitute the majority of the neonitmal hyperplasia region.
In general, glucose uptake is retained in normal VSMCs 17 and is increased by inflammatory stimuli 18 . As expected, F-18 FDG PET imaging and autoradiography revealed prominent uptake of FDG in the right carotid artery of atherosclerotic rats. However, FDG uptake was not observed in the right carotid artery of normal rats. Interestingly, FDG distribution appeared to be stratified in the atherosclerotic right carotid artery, with higher FDG uptake in the inner circular layer compared to the surrounding outer layer. These findings were further supported by the increased expression of GLUT1 in synthetic VSMCs of neointima region. Although further studies are warranted to clarify the mechanisms underlying this stratified distribution, we hypothesize that the synthetic activity of VSMCs may be greater in the early phase of neointimal hyperplasia than in the later phases.
F-18 FDG PET is a potentially useful tool for evaluating the activity of synthetic VSMCs in a wide range of vascular remodeling diseases. In atherosclerosis, synthetic VSMCs induce neointimal hyperplasia, narrow the lumen, and provide substrates for lipoprotein retention, thereby accelerating the progression of atherosclerosis 19 . In addition to synthetic VSMCs, inflammatory responses also play a key role in vascular wall damage in atherosclerosis 20 . In particular, macrophages appear to significantly contribute to foam cell formation and plaque rupture 20 . While neointimal hyperplasia is also formed in in-stent restenosis and transplant vasculopathy, inflammatory responses appear to play only a minor role in these vasculopathies 19 . Recently, Kim et al. 21 reported that 68 Ga-labeled NOTA-neomannosylated human serum albumin (MSA) could target macrophages in atherosclerosis. Using a combination of FDG and MSA, the molecular basis of synthetic VSMC and macrophage activities in atherosclerotic lesions could be easily obtained.

Conclusion
The activity of synthetic VSMCs was successfully visualized by F-18 FDG PET. Therefore, F-18 FDG PET is a promising noninvasive imaging modality for evaluating synthetic VSMCs, especially in vascular remodeling disorders.

Materials and Methods
Animals. Eight male Sprague-Dawley (SD) rats (7 weeks old, 200 g body weight) were purchased from Orient-Bio (Seongnam, Korea). 4 rats were enrolled in atherosclerotic group and 4 rats were in normal control group. All rats were maintained under a 12-h/12-h day/night cycle with ad libitum water and meals. All experimental protocols and procedures were approved by the Ethics Committee and the Institutional Animal Care and Use Committee of Korea University College of Medicine (Approval No. KOREA-2016-0041). All experiments were performed in accordance with the approved guidelines and regulations.

Induction of atherosclerosis.
After a 1-week adaptation period, right partial carotid artery ligation was performed as previously described 9 . Briefly, anesthesia was induced with 3.5% isoflurane with a 2:1 N 2 O/O 2 mixture in a vented anesthesia chamber and then maintained by the administration of 2 to 2.5% isoflurane with a 2:1 N 2 O/O 2 mixture through a nasal cone. The right external carotid artery, right internal carotid artery, and right occipital artery were ligated with a 6-0 silk suture. After ligation, vitamin D3 (6 × 10 5 IU/kg) was intraperitoneally injected for 2 consecutive days 22 , with the exception of the normal control group. Rebsamen et al. 23 reported that vitamin D3 facilitates VSMC migration in the SD rat aorta. The atherosclerotic group was fed daily with a commercially available atherogenic diet (D12336, Research Diets, NJ, USA) for 4 weeks. The normal group underwent a sham operation without ligation of carotid arteries or vitamin D3 injections. Normal rats were fed a diet of normal chow for 4 weeks.

F-FDG PET imaging and analysis.
Images were acquired using a small animal PET/CT (computed tomography) scanner (eXplore Vista DR PET/CT, GE Healthcare, Milwaukee, WI, USA). Rats were not fasted before examination and were anesthetized with 2% isoflurane at 1 L/min oxygen flow during the PET/CT scan. Rats were placed in the prone position under the scanner. PET image acquisition started 40 min after the injection of 37 MBq/0.2 ml of F-18 FDG via the tail vein. Static PET scans were acquired for 20 min in a single bed position covering the carotid artery region. The axial field of view (FOV) of the PET scanner was 48 mm in length. CT scanning (40 kV, 250 µA) was initiated after PET scanning of the same area. Images were reconstructed using Fourier rebinning and the ordered subsets expectation maximization (OSEM) algorithm with decay, attenuation, random, and normalization corrections. The voxel size was 0.3875 × 0.3875 mm; axial slice thickness was 0.775 mm. Maximum intensity projection (MIP) images, axial views, sagittal views, and fusion images were processed after reconstruction. Following image reconstruction, image analysis was performed with A Medical Image Data Examiner Software (AMIDE, version 1.0.4) 24 . On each image, regions of interest (ROI) were drawn over the carotid artery region and the standardized uptake values (SUVs) were measured. The SUV was calculated as activity concentration (ROI; MBq/ml)/injected dose (MBq)/total body weight (g).
Autoradiography. Immediately after completion of PET/CT scanning, the carotid arteries were harvested, fixed in 4% paraformaldehyde (PFA), and mounted on glass slides. The prepared tissues were exposed to imaging plates and images were acquired using a BAS-200 system (FLA-2000, FUJIFILM, Tokyo, Japan).

Immunohistochemistry.
Harvested carotid arteries were fixed with 4% PFA and preserved in 30% sucrose solution. Tissues were embedded in Optimal Cutting Temperature (OCT) compound (Scigen Scientific, Gardena, CA, USA). Axial sections of 4-μm thickness were cut using a cryostat microtome (Leica CM 3050 S, Leica  USA) were used as secondary antibodies. Cell nuclei were counterstained with DAPI. Histopathologic evaluation was performed by staining with hematoxylin and eosin. All images were acquired on a confocal microscope (LSM800, Carl Zeiss, Oberkochen, Germany) or an upright light microscope (BX51, Olympus, Tokyo, Japan).
Statistical analysis. The Mann-Whitney U test was used as a statistical method using SPSS software version 17.0 (SPSS Inc, Chicago, IL, USA). A p-value < 0.05 was defined as statistically significant.