In vivo MRI detection of atherosclerosis in ApoE-deficient mice by using tenascin-C-targeted USPIO

Abstract

Background

Atherosclerosis is the main cause of cardiovascular and cerebrovascular diseases. Non-invasive molecular imaging to detect and characterize the plaques is essential for reducing life-threatening cardiovascular events.

Purpose

To investigate the possibility of the anti-tenascin-C-USPIO specific probe as a molecular marker of atherosclerotic plaques detected by 7.0-T magnetic resonance imaging (MRI).

Material and Methods

Twenty ApoE^-/- mice fed with a high fat diet were used for detecting the aorta arch atherosclerotic plaques by 7.0-T MRI at 16 and 24 weeks. Ten mice in the targeted group were injected with anti-tenascin-C-USPIO and another ten in the control group were injected with pure USPIO (n = 5 each time point in each group). Histopathologic examination was used to evaluate the plaques and immunohistochemistry analysis was used to compare tenascin-C expression.

Results

The relative signal intensity (rSI) changes of the targeted group decreased more than those of the control group (16 weeks: −15.65 ± 0.78% vs. −3.43 ± 2.57%; 24 weeks: −26.38 ± 1.54% vs. −11.12 ± 1.60%, respectively; P < 0.05). Histopathological analyses demonstrated visible atherosclerotic plaques formation and development over time from 16 weeks to 24 weeks. Tenascin-C expression of the plaques at 24 weeks was higher than that at 16 weeks (0.22 ± 0.04 vs. 0.13 ± 0.02, P < 0.05). The MR images correlated well with the progression of atherosclerotic plaques.

Conclusion

Tenascin-C expression increased with the progression of atherosclerosis. Anti-tenascin-C-USPIO could provide a useful molecular imaging tool for detecting and monitoring atherosclerotic plaques by MRI.

Keywords

Tenascin-C atherosclerosis USPIO magnetic resonance image

Introduction

Atherosclerosis, which is the pathological basis of cardiovascular and cerebrovascular diseases, has seriously threatened human health (1,2). Non-invasive imaging modalities to detect atherosclerotic plaques and monitor disease progression is essential for reducing the occurrence of acute cardiovascular diseases. Conventional imaging techniques are either invasive or unable to assess the disease accurately (3 –5). These examinations mainly give insight into plaque morphology but cannot effectively predict plaque biological changes. Molecular imaging is promising as a noninvasive modality to detect and characterize atherosclerotic plaques because it lacks ionizing radiation and can measure molecular compositions in the plaques at the molecular level (6,7). Therefore, molecular magnetic resonance imaging (MRI) for evaluating atherosclerosis would be needed.

Tenascin-C, a multifunctional extracellular glycoprotein, plays a crucial role in the formation and progression of atherosclerosis in both animals and humans (8 –10). Several studies show tenascin-C is highly expressed in the advanced atherosclerotic plaques and its high expression is related to inflammatory change and plaque rupture. Tenascin-C serves as an effector of tumor necrosis factor alpha (TNF-α) in the formation of foam cell and atherosclerosis through Toll-like receptor 4 (TLR-4) (11). Additionally, matrix metalloproteinase-9 (MMP-9) protein and messenger RNA (mRNA) expression in macrophages could be upregulated by tenascin-C and then be activated (8). The activated MMP-9 would promote the extracellular matrix degradation and accelerate plaque progression. In advanced atherosclerotic plaques, tenascin-C degradation products induce smooth muscle cell (SMC) apoptosis and finally lead to plaque rupture (12). All these evidences show that tenascin-C is one of the most important proteins related to atherosclerosis.

In the present study, ultra-small superparamagnetic iron oxide (USPIO) nanoparticles were labeled with the mentioned monoclonal anti-mouse tenascin-C antibody to synthesize a novel tenascin-C-targeted MR molecular probe (anti-tenascin-C-USPIO). The aim was to detect aorta arch atherosclerotic plaques and evaluate plaque progression in ApoE^-/- mice by MRI.

Material and Methods

Animal model of atherosclerosis

All animal experimental procedures complied with the institutional guidelines and were approved by the local Animal Ethics Committee. Twenty male ApoE^-/- mice (C57BL/6 J background, 21.7 ± 0.7 g) were purchased from Viral River Laboratory (Beijing, PR China). All the mice were fed with a high fat diet (21% fat, 0.15% cholesterol; Western diet) at the age of eight weeks.

All the mice were classified into targeted probe group and control group. The MRI protocol was performed at two time points (16 and 24 weeks) between the two groups (n = 5 each time point in each group). A MRI scan was performed before the USPIO agent administration as the baseline imaging and a second MRI scan was obtained 24 h post contrast.

Synthesis of anti-tenascin-C-USPIO

Initially, 2 mg PEG-coated USPIO (588 µL) was diluted in 412 µL Tris (hydroxymethyl) aminomethane buffer (pH 7.2, 25 mM). Then 1 mg of 1-Ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCL) and 2.8 mg of sulfo-N-hydrosuccinimide (Sulfo-NHS) were added to the particle solution and shaken up on the oscillator for 15 min. After completing the reaction, the solution was ultrafiltered with a centrifugal filter device (300 Kd, PALL, Port Washington, NY, USA) to remove excess EDC.HCL and Sulfo-NHS. Then 100 µL of anti-mouse tenascin-C antibodies was added and stirred for 2 h at 37℃. Finally, the conjugated USPIO was purified with phosphate buffered saline (PBS) and stored in PBS (0.1 M, pH 7.4) at 4℃ (13,14).

Characteristics of anti-tenascin-C-USPIO

The morphology of the USPIO was observed by transmission electron microscope (TEM) (JEOL-100CX, Tokyo, Japan). The size and distribution of the particles were measured by Image-Pro Plus 6.0. Zeta potential was measured by instruments (Nano ZS, Malvern, UK). The hydrated particle sizes were measured by dynamic light scattering (DLS) (Nano ZS, Malvern, UK). T2 relaxation rate was performed using a 3.0-T MRI scanner (Verio Tim; Siemens, Erlangen, Germany); anti-tenascin-C-USPIO was diluted to the following concentration gradients: 0.1 mmol/L; 0.2 mmol/L; 0.3 mmol/L; 0.4 mmol/L; 0.5 mmol/L; and 0.6 mmol/L. T2 relaxation rate was calculated as 1/T2. The targeted USPIO specificity was evaluated by a tenascin-C mouse enzyme-linked immunosorbent assay (ELISA) kit (R&D system). In ELISA analysis, boiled targeted USPIO and pure USPIO were used as controls.

MR protocol

In vivo MRI was performed using a 7.0-T MRI scanner with a small animal coil and mouse cradle. Double cardiac and respiratory gated devices were connected. Three patch electrodes were placed on the mice’s paws to collect the electrocardiogram (ECG) signal and a pressure sensor was placed under the abdomen for the respiratory signal. The mice were initially anesthetized by 4% isoflurane/gas mixture and were maintained on anesthesia with 1.5% isoflurane/gas mixture during scanning.

Mice in the targeted probe group were injected with anti-tenascin-C-USPIO via tail vein at a dose of 10 mg Fe/kg body weight (13), while mice in the control group were injected with the same dose of pure USPIO. A bright-blood cine-mode FLASH sequence was used for aortic arch localization and a quick check of the position of the animal, performed on seven transverse joined slices centered on the base of the heart. These reference slices were acquired with a gradient echo (GE) sequence. Based on these overview scans, a proton density weighted dual-echo multi-slice multi-echo (MSME) sequence can be planned and then obtained in the transverse plane. The parameters of this sequence were as follows: TR = 1891.5 ms; TE = 65 ms/13 ms; field of view (FOV) = 2.0 × 2.0 cm; matrix = 256 × 256; slice thickness = 0.7 mm; number of averages (NA) = 2.

Image analysis

Two radiologists with six and five years of experience, respectively, blinded to the USPIO types and histological results, drew regions of interest (ROIs) manually over the atherosclerotic plaque by using the Image J analysis software. Relative signal intensity (rSI) was defined as plaque SI divided by muscle SI in the same slice. The percent of normalized enhancement (% NENH) was calculated using the following equations: % NENH = (rSI_post−rSI_pre)/rSI_pre × 100% (13), where rSI_post represents rSI value after contrast agent administration, and rSI_pre represents pre-contrast rSI. The measurement results of the two radiologists were used to assess the inter-reader reproducibility. To assess the intra-reader reproducibility, the first reader reassessed all the images one month after the first measurement. The average results from the first reader were used for further statistical analysis.

Histopathology and immunohistochemistry

After MRI scanning, the mice were anesthetized by 10% chloral hydrate. A tiny slit was cut in the right atrial appendage and 50 mL PBS was slowly injected into the left ventricle for cardiac perfusion. Subsequently, 10 mL or more of 4% paraformaldehyde solution was injected. After finishing perfusion, the thoracic aorta was harvested and fixed in 4% paraformaldehyde solution. The specimens were embedded in paraffin and serially cut with a 3 µm thickness every 50 µm and approximately 50 sections were obtained per vessel. Hematoxylin and eosin (H&E) stain was performed to identify the presence of atherosclerotic lesions. In pathology, atherosclerotic lesions could be divided into three categories: (i) early lesions; (ii) progressive (advanced) lesions; and (iii) complicated lesions (15). The criteria were defined as follows: intimal thickening and foamy cell accumulation were considered as early lesions, progressive lesions included inflammatory cell infiltrations (foamy cells, leukocytes, and others), while complicated lesions accompanied by secondary lesions, such as calcification or stenosis. For immunohistochemical analysis, sections were stained with rat anti-mouse-tenascin-C mAb (1:100, Invitrogen, Carlsbad, CA, USA) and anti-mouse-CD68 mAb (1:100, Abcam, Cambridge, UK), a macrophage marker. The expression and distribution of tenascin-C and CD68 in the plaques were observed by light microscope. The positive stained area percentage (PSAP) (positive reaction area/total measurement area) calculated by Image-Pro Plus 6.0 represented tenascin-C expression level. The specific method was as follows: five sections of each sample in two random sights were selected to conduct the semi-quantitative analysis of PSAP and the average results were used for further analysis. Prussian blue stain was used to prove the presence of USPIO.

Statistical analysis

All data were expressed as mean ± standard deviation (SD). Quantitative data were analyzed using unpaired t test. Inter- and intra-reader reproducibility was evaluated by using the intra-class correlation coefficient (ICC) with 95% confidence intervals (CI). The ICC was in the range of 0–1.00 and the values were interpreted as follows: r < 0.40 = poor; 0.41–0.60 = moderate; 0.61–0.80 = good; ≥ 0.81 = excellent. P < 0.05 was considered to represent statistically significant differences. Statistical analyses were performed by using SPSS version 21.0 (SPSS, IBM, Chicago, IL, USA).

Results

Characterizations of anti-tenascin-C-USPIO

The anti-tenascin-C-USPIO solution is black-brown in color and shows apparent magnetic properties when it is close to the magnet. TEM image demonstrates the probe is spherical or spheroidal in shape and well distributed in PBS solution (Fig. 1a). The hydrated diameter of the probe was greater than that of untargeted USPIO (47.18 ± 1.48 nm vs. 33.53 ± 1.16 nm), indicating that the conjunction was successful. The Zeta potential distribution was −13.8 mV. T2 relaxivity was 168.24 mM^–1 s^–1 (Fig. 1b and c). The ELISA results showed anti-tenascin-C-USPIO maintained high biological activity whereas the pure USPIO and boiled anti-tenascin-C-USPIO showed negative activity (Fig. 1d).

Fig. 1.

(a) TEM image shows the targeted probe is well dispersed in PBS solution. Upper right insert shows apparent magnetism of the probe liquid. (b, c) T2 relaxation rate of the probe under different concentrations of Fe and T2 relaxivity. (d) ELISA results of different nanoparticles. Data are expressed as mean ± SEM (n = 5). *P < 0.05.

In vivo MRI

Fig. 2 shows the comparison of signal loss between the two groups at two time points. The rSI changes in the targeted probe group (–15.65 ± 0.78% and −26.38 ±1.54%) showed significant differences compared to the control group (–3.43 ± 2.57% and −11.12 ± 1.60%; P < 0.05) at 16 and 24 weeks. In the targeted USPIO group, rSI decreased more at 24 weeks than 16 weeks (P < 0.05). Fig. 3 shows the representative MR images of atherosclerotic plaques between the targeted group and control group. The presence of USPIO was verified by Prussian blue staining. More USPIO deposition was found in the targeted group than that in control group.

Fig. 2.

The comparisons of NENH% between targeted probe group and control group are shown. *P < 0.05.

Fig. 3.

Representative MR images of aorta arch atherosclerotic plaques in ApoE^-/- mice before and 24 h after administration of targeted USPIO and pure USPIO (a, b). The red arrows indicate atherosclerotic plaques. Prussian blue staining to verify the existence of USPIO in the plaques (×200). The black arrows indicate the Fe depositions. (a) targeted group; (b) control group.

Histopathologic analyses demonstrated visible atherosclerotic plaques formation and development over time from 16 weeks to 24 weeks. Many progressive and complicated lesions were observed microscopically. Immunohistochemical results showed tenascin-C and CD68 co-expressed in macrophage-rich sites, which were mainly expressed in and around the lipid core (Fig. 4). And tenascin-C expression of atherosclerotic plaques at 24 weeks was higher than that at 16 weeks (0.22 ± 0.04 vs. 0.13 ± 0.02, P < 0.05, Fig. 5). The MR images correlated well with the progression of atherosclerotic plaques.

Fig. 4.

Histopathological and immunohistochemical analyses of the atherosclerotic lesions in ApoE^-/- mice. The plaques of aorta arch were stained for tenascin-C and CD68 (×100).

Fig. 5.

The comparison of tenascin-C expression in the plaques at 16 weeks and 24 weeks is shown. *P < 0.05.

Measurements reproducibility

Good inter-reader agreements and excellent intra-reader agreements were obtained from the SI measurements, with ICCs values of 0.871 (95% CI = 0.769–0.929) and 0.935 (95% CI = 0.878–0.966), respectively.

Discussion

The present study demonstrated that tenascin-C expression increased with the progression of atherosclerosis, and the synthesized molecular probe anti-tenascin-C-USPIO could provide a useful molecular imaging tool for detecting and monitoring atherosclerotic plaques.

Previous studies have indicated that it is possible to characterize and monitor atherosclerosis by using MRI contrast agent USPIO (16 –18). The PEG-coated iron oxides are known to reduce plasma protein binding, delay clearance by the reticuloendothelial system and increase particle circulation times (19). These characteristics make it possible for the targeted iron oxide nanoparticles to reach the tissue of interest. Similar to the abovementioned properties, our constructed probe anti-tenascin-C-USPIO was able to accumulate in the atherosclerotic plaques and to produce prominent signal decrease for effective molecular MRI.

The principle for tenascin-C targeted USPIO used in this study was that tenascin-C was highly expressed in advanced atherosclerotic plaques. Several studies indicated that in human coronary plaques, tenascin-C was highly expressed around the lipid core, plaque shoulder, and ruptured places (12,20). Also, in patients with acute coronary syndrome, serum tenascin-C level was significantly high (21). Our study showed similar results as previous studies. Histopathologic analyses in the current study demonstrated the progression of the plaque lesions in the aortic arch from 16 to 24 weeks and increasing tenascin-C staining was observed in the lesions. In addition, MR images of atherosclerotic plaques correlated well with the histopathological results. All these findings have highlighted that tenascin-C plays an important role in atherosclerotic disease and could be selected as an appropriate molecular probe.

At present, a variety of probes and markers were constructed to detect the atherosclerotic plaques and evaluate plaque progression. Bigalke et al. used ⁶⁴Cu-labeled CD68-Fc to detect the lipid-rich plaques (22). Ishino et al. indicated the potential of ⁹⁹mTc-LOX-1-mAb for targeting LOX-1 and evaluating the vulnerability of atherosclerotic lesions (23). Human antibody against the C domain of tenascin-C was used to visualize atherosclerotic plaques ex vivo (24). However, low spatial resolution and ionizing radiation limited its wide application. It is well-known to us that atherosclerosis is a chronic inflammatory process, and inflammatory cells play a central role on this process. On the basis of this theory, some studies targeted macrophages based on gadolinium agents to detect vulnerable plaques in ApoE^-/- mice in vivo (25,26). Some other studies have targeted matrix metalloproteinases (MMPs) (27), activated platelet (28), and oxidized low-density lipoproteins (29). The majority of these probes are based on Gd-DOTA contrast agents and mainly focused on just one period of atherosclerotic plaques. In our present study, we synthesized the probe with MRI agent USPIO, which is small in size and suitable for vascular imaging. Besides that, compared with Gd-containing micelles, iron oxide particles were less toxic. Different periods were performed so that signal intensity changes can be observed dynamically. Signal loss was more obvious in the probe group than the control group, and signal changes of plaques decreased more at 24 weeks than those at 16 weeks. The reason was that, in the atherosclerotic plaques, a large number of macrophage cells accumulated, and these inflammatory cells accelerated the plaques progression. Macrophage is known to be one of the sources of tenascin-C (8). Immunohistochemistry confirms the colocalization of tenascin-C and CD68 expression and iron oxide nanoparticles deposition. These results indicated that the constructed probe could reflect the plaque biology and was considered as a promising target for molecular MRI of atherosclerosis. Thus, we have provided another targeted molecular imaging of USPIO-based contrast agents for detecting and monitoring the progression of the plaque lesions.

Some limitations within present study should be noted. First, the selection of imaging time was 24 h after intravenous injection, and although some studies indicated 24 h is a suitable time period (13,30), different time periods are needed. Second, the tail vein injection may allow the iron particles to be swallowed by the monocyte phagocytic system, to a certain extent affecting the amount of probes binding. How to reduce this non-specific uptake needs to be studied in further study.

In conclusion, the anti-tenascin-C-USPIO probe provides important information for characterizing atherosclerotic plaques and may evolve into a clinically relevant non-invasive imaging tool for detecting and characterizing plaques in vivo.

Footnotes

Acknowledgments

The authors thank Beijing Onerder Hightech. Co. Ltd. for its probe synthesis assistance during the experiment.

Declaration of Conflicting Interests

The author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Contract grant sponsor: the National Natural Science Foundation of China (contract grant no. 81471723).

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