99mTc-labeled bevacizumab for detecting atherosclerotic plaque linked to plaque neovascularization and monitoring antiangiogenic effects of atorvastatin treatment in ApoE−/− mice

Atherosclerotic neovascularization plays a significant role in plaque instability as it provides additional lipids and inflammatory mediators to lesions, and resulting in intraplaque hemorrhage. Vascular endothelial growth factor-A (VEGF-A) is considered the predominant proangiogenic factor in angiogenesis. Bevacizumab, a humanized monoclonal antibody, specifically binds to all VEGF-A isoforms with high affinity. Therefore, in this study, we designed 99mTc-MAG3-bevacizumab as a probe, and then investigated its usefulness as a new imaging agent for the detection of plaque neovessels, while also assessing the therapeutic effect of atorvastatin treatment. The ApoE−/− mice treated with atorvastatin were used as the treatment group, and C57BL/6 J mice were selected as the control group. 99mTc-MAG3-bevacizumab uptake was visualized on atherosclerotic lesions by non-invasive in-vivo micro-SPECT/CT and ex-vivo BSGI planar imaging. The value of P/B in each part of the aorta of ApoE−/− mice was higher than in the treatment group and the C57BL/6 J mice, which was confirmed by Oil Red O staining, CD31 staining and VEGF immunohistochemistry staining. 99mTc-MAG3-bevacizumab imaging allowed for the non-invasive diagnosis and assessment of plaque neovascularization. Furthermore, this probe may be used as a new molecular imaging agent to assess the antiangiogenic effect of atorvastatin.

Although a variety of factors have been found to contribute to the process of angiogenesis, vascular endothelial growth factor-A (VEGF-A) is recognized as the predominant proangiogenic factor 9,10 . Prior studies have demonstrated that VEGF-A upregulation results in an increased permeability of vascular endothelial cell which caused immature microvessels 11,12 . VEGF-A, the main proangiogenic isoform of the family, binds primarily to VEGF receptor (VEGFR)-1 and VEFGR-2 13 . Thus, molecular imaging of the components of VEGF-A or VEGFR should indicate the angiogenic process of plaques.
In vivo imaging of VEGFRs may be achieved by radiolabeled VEGF-A, but each different VEGF isoform has a different affinity for VEGFR-1 and VEGF-2, suggesting a potential role for VEGF-based imaging for neovascularization 14,15 . Bevacizumab, a humanized monoclonal antibody, specifically binds to all VEGF-A isoforms with high affinity, and inhibits its interaction with VEGFR-1 and VEGFR-2, which is currently used in the clinic for cancer treatment and has been approved by the Food and Drug Administration (FDA) [16][17][18][19] . Several studies have used bevacizumab to target nuclear medicine probes by 89 Zr,111 In and 99m Tc for labeling of tumor angiogenesis assessment [20][21][22][23][24][25] . To date, however, this probe of bevacizumab has not been used for plaque neovascularization in vivo. Golestani et al. 23 used 89 Zr-bevacizumab ex vivo imaging to evaluate excised carotid artery atherosclerotic plaque, and the results showed both that 89 Zr-bevacizumab uptake was obviously correlated with VEGF immunohistochemical staining scores, and also that it is possible to detect VEGF using 89 Zr-bevacizumab PET. Furthermore, both 89 Zr and 111 In are cyclotron produced with their own limitations. 99m Tc is the most widely used diagnostic radionuclide in nuclear medicine, as it has attractive nuclear properties and easy accessibility from a 99 Mo/ 99m Tc generator system. Therefore, in this study, we designed and prepared 99m Tc-labeled bevacizumab as a probe for plaque neovascularization imaging in an ApoE −/− atherosclerotic mice model. In addition, previous studies have shown that treatment with stains for atherosclerosis (AS) can also exert antiangiogenic effects and reduce intraplaque neovascularization. Hence, we further investigated its usefulness as a new imaging agent to assess the therapeutic effect of atorvastatin, and to verify its antiangiogenic effects by in vivo molecular imaging.
After SPECT/CT imaging, we immediately took out the aorta and performed ex vivo planar imaging by breast specific gamma imaging (BSGI) for the excised aortas. Figure 5c illustrates the gross morphology of the aortic plaque, which confirms the SPECT/CT and corresponding BSGI imaging finding that the more plaque in an area of the aorta, the more tracer uptake in that area. In addition, 99m Tc-MAG 3 -bevacizumab uptake in ApoE −/− mice was higher than in the treatment and control groups. The therapeutic group was also higher than the C57BL/6 J group by visual analysis. And the same semi-quantitative analysis of mean value of plaque to non-plaque counts ratio (P/N), ApoE −/− versus treated group (P/N: 3.50 ± 0.20 versus 2.26 ± 0.40, P = 0.0085, t = 4.83), ApoE −/− versus C57BL/6 J group (P/N: 3.50 ± 0.20 versus 1.40 ± 0.11, P < 0.0001, t = 16.06), treated versus C57BL/6 J group (P = 0.0232, t = 3.58), shown in Fig. 5d. Oil Red O staining. Next, Oil Red O staining was performed on all mice groups to compare the percentage of plaque areas in both the total aorta and specific parts of the aorta, as shown in Fig. 6. The ApoE −/− mice presented with a significantly higher percentage area of aortic lipid plaque than did the treated mice (43.24 ± 6.13% vs. 22.67 ± 4.53%; P = 0.0017, t = 5.40), as shown in Fig. 6a and b. There was no typical plaque was observed in the aorta of C57BL/6 J mice. Furthermore, the percentage area of plaque in the aortic arch and abdominal aorta in ApoE −/− mice were both higher than treated mice, with statistical significance (P = 0.0007, t = 6.37) and (P = 0.0037, t = 4.60). The difference in percentage area of plaque in the thoracic area, however, was not statistically significant (P > 0.05) (Fig. 6c) between the groups.  The aorta in-vivo SPECT/CT imaging and pathology. HE and Masson trichrome staining and CD31 and VEGF immunohistochemistry staining were performed on the aorta sections sliced at points with obvious radioactivity in SPECT/CT images. The lesions with accumulated radioactivity were consistent with the anatomical structure of the plaques. Interestingly, the thoracic aorta with the most radioactivity showed the most distinct plaque in corresponding areas (Fig. 8a,b). Figure 8e and f further illustrate that the level of 99m Tc-MAG 3 -bevacizumab uptake in plaque was primarily related to the numbers of new blood vessels in the plaque on pathological results.

Discussion
To our current knowledge, plaque neovascularization is recognized as a critical process of the progression of atherosclerotic plaques and is related to plaque vulnerability. Histopathological analysis has been the gold standard for visualizing newly formed vessels in unstable lesions, though it has the disadvantage of requiring tissue samples. Functional or molecular imaging techniques, however, have demonstrated distinct visualization of plaque microvessels non-invasively. Bevacizumab, a humanized monoclonal antibody against VEGF-A with reduced immunogenicity, is currently approved by the FDA to treat some cancers, inhibiting tumor angiogenesis in the clinic setting. However, it also shows serious adverse effects, including cardiovascular diseases, such as hypertension and thromboembolism, especially for the cancer patient with pre-existing arterial cardiovascular disease [26][27][28] . For that reason, screening for tumor patients with a high risk for atherothrombotic disease is crucial to avoid the serious complications of bevacizumab treatment. Bevacizumab radionuclide imaging, as a noninvasive method, not only could be quite helpful in guiding bevacizumab therapy for many cancers because of interpatient tumor heterogeneity in tumor imaging aspect, maybe but may also discourage some tumor patients with unstable plaque from receiving bevacizumab therapy to avoid the serious complications in atherosclerotic imaging aspect. and C57BL/6 J mice. "*", "**" and "***" indicated P < 0.05, < 0.01 and < 0.001, respectively; while "ns" denoted no significance.  Therefore, we applied 99m Tc-MAG 3 -bevacizumab as a probe for atherosclerotic imaging to reflect microvessels of plaque linked to plaque instability in this study.  The study presented an indirect method for 99m Tc labeling bevacizumab, applied NHS-MAG 3 as a bifunctional chelator, which yielded good radiolabeling yields that reached over 80%, similar to 99m Tc carbonyl DTPA-bevacizumab reported by the Kameswaran M, et al. 25 . The immunoreactivity results confirmed that bevacizumab still kept its reactivity to the VEGF receptor after labeling with 99m Tc. The biodistribution pattern of 99m Tc-MAG 3 -bevacizumab in wild-type C57BL/6 J mice indicated a high radioactivity in the blood over the times observed, which could be because of bevacizumab with a relatively long biological half-life of approximately 3 weeks. The fact that the probe was eliminated through both the kidneys and the hepatobiliary system may be a reason for the high accumulations of radioactivity in those organs. The high radioactivity in these organs is consistent with previous studies that showed biodistribution of both probes 99m Tc(CO) 3

-bevacizumab and 99m
Tc-carbonyl-DTPA-bevacizumab 25,29 . The aortic artery and heart with relatively low 99m Tc-MAG 3 -bevacizumab uptake may illustrate the potential value for AS imaging. Unfortunately, the probe showed relatively slow clearance rates in pharmacokinetic experiments, and this rate will have resulted in a low signal to noise ratio (SNR) for diagnostic imaging. These findings are similar to those in previous studies that fabricated these probes of 99m Tc-carbonyl-DTPA-bevacizumab, 124 I-iodinated-VG76e and 123 I-VEGF for imaging VEGF in vivo 25,30,31 . Thus, new molecular probes need to address this shortcoming in our study later.
In our experiments, ApoE −/− , treated mice and C57BL/6 J models were established, and the level of serum lipid analyses revealed obvious higher in ApoE −/− than in C57BL/6 J mice in TC and LDL, but treated group without markedly decreasing than ApoE −/− mice, just as the previous studies showed that 10 mg/kg/d atorvastatin treatment did not lower plasma total cholesterol levels [32][33][34] . And the protective index of serum HDL showed remarkably difference among intergroup. The classical pro-inflammatory cytokines (IL-1β and CRP) were significantly increased in ApoE −/− mice, compared to C57BL/6 J mice, especially the IL-1β, which played a central role in regulating immune, and inflammation response. Compared with ApoE −/− mice, the treated group with 10 mg/ kg/d atorvastatin displayed the decreased serum IL-1β and CRP levels. So, our study demonstrated the beneficial effect of atorvastatin is downregulated inflammatory cytokines with the inhibition of macrophage infiltration, in accord with the previous reach performed by Nie P, et al. 32 . The role of TGF-β in atherosclerotic progression is controversial, and studies largely supported an anti-atherogenic role of TGF-β 34 , with revealing a negative correlation between plasma TGF-β level and the extent of AS, then our study also confirmed this conclusion that the level of plasma TGF-β was significantly lower in ApoE −/− mice and therapy mice than C57BL/6 J mice.
There were few studies used bevacizumab as a probe to image plaque neovasculature, just Golestani et al. 23 displayed the results that 89 Zr-bevacizumab uptake in the excised carotid artery atherosclerotic plaques for ex vivo imaging, was obvious correlated with VEGF immunohistochemical staining scores. In our study, the results showed that in vivo 99m Tc-MAG 3 -bevacizumab micro SPECT/CT imaging was allowed noninvasive visualization of atherosclerotic plaques, and the tracer uptake was higher in ApoE −/− than C57BL/6 J group by visual and semi-quantitative analysis. The results of ex-vivo planar view was just similar to SPECT/CT imaging that obvious radioactivity uptake in ApoE −/− not in C57BL/6 J group both visual and semi-quantitative analysis, and was direct reflected the tracer uptake in atherosclerotic plaques because of it avoiding interference from the surrounding tissue with high radioactivity accumulation. 99m Tc-MAG 3 -bevacizumab concentration in aortic plaques in-vivo and ex-vivo imaging was further confirmed by gross morphology, Oil Red O staining, CD31 and VEGF immunohistochemistry staining of plaque. Oil Red O staining was used to assess lipid deposition with arterial lumina 35 . As for the immunohistochemistry, it was the gold standard to look into neovascularization in plaques. Our study indicates that 99m Tc-MAG3-bevacizumab could be an available probe for atherosclerotic angiogenesis imaging, non-invasively, and evaluate neovascularization of plaques that tightly linked to plaque rupture.
It is well known that atorvastatin as HMG-CoA reductase is broadly applied in lipid-lowering treatment, and some research had shown it also with pleiotropic effects, which included anti-angiogenic effects 32,33,36,37 . Roth et al. 36 reported that atorvastatin treatment was responsible for reduction in the expression of inflammatory cytokines, coronary stenosis, and the occurrence of intraplque microvessels. Our results also had proved atorvastatin therapy with pleiotropic effects in decreasing the level of plasma inflammatory factors, plaques area of aorta and stenosis of arterial lumen which confirmed by Oil Red O staining, HE and Masson staining, as well as atherosclerotic neovascularization which affirmed by immunohistochemistry.
It has been presented in previous studies that molecular imaging with targeted MRI, nuclear medicine and ultrasound probes can be applied for assessing the effect of stains treatment in AS, but most were to assess the anti-inflammatory treatment of stains [38][39][40] . In this study, 99m Tc-MAG3-bevacizumab as SPECT probes were used for assessing the anti-angiogenic effects for atorvastatin therapy in AS model mice. Our results showed that the treatment group with 99m Tc-MAG 3 -bevacizumab uptake was lower than that of ApoE −/− mice in in-vivo SPECT/CT imaging by visual analysis, and ex-vivo BSGI imaging both visual and semi-quantitative analysis, and these imaging results were consistent with pathological analysis. This study revealed the potential of 99m Tc-MAG 3 -bevacizumab as a molecular imaging probe to non-invasively assess the effects of antiangiogenic.
In summary, this study demonstrated that a 99m Tc-MAG 3 -bevacizumab targeted SPECT probe allowed for non-invasively diagnosing and assessing the neoangiogenesis of atherosclerotic plaques in ApoE −/− mice. It is a potential probe to not only guide bevacizumab therapy because of interpatient heterogeneity, but also to screen suitable patients with bevacizumab treatment to avoid serious complications in cardiovascular diseases. Furthermore, this probe as a new molecular imaging agent could be used to assess the antiangiogenic effect of atorvastatin.

Methods
Approvals. The experimental protocols were approved by the medical ethics of committee Zhongshan Hospital, Fudan University.
The process of 99m Tc-MAG 3 -bevacizumab synthesis was as follows: we added 100 μg bevacizumab in 0.30 M HEPES to a tube containing NHS-MAG 3 powder, and then incubated the mixture at room temperature for 1 hour with occasional agitation. The mixture was purified by the centrifugation method to remove unconjugated and hydrolyzed MAG 3 using 0.25 M ammonium acetate as eluent, to obtain MAG 3 -bevacizumab. Then, MAG 3 -bevacizumab conjugate was added to a combined solution of 45 μL ammonium acetate (0.25 M) and 15 μL tartrate buffer, with no more than 25 μL (around 37MBq) 99m TcO 4 − . Immediately, 4 μL of freshly prepared 1 mg/ mL SnCl 2 •2H 2 O solution were added. After gently vortexing for a short time, the combined solution was incubated at room temperature for 1 hour. The purification of 99m Tc-MAG 3 -bevacizumab was by centrifugation, and its radiochemical purity was measured by a size-exclusion HPLC shift assay (Agilent Technologies, 1260 Infinity).
In vitro stability, pharmacokinetics and biodistribution of 99m Tc-MAG 3 -bevacizumab. The in vitro stability in 0.9% NaCl, 0.05 M PBS and serum was evaluated at 1, 2, 4, and 6 h at room temperature and 37 °C. Male wild-type C57BL/6 J (n = 20) mice with the age of 8 weeks were injected via the caudal vein with 99m Tc-MAG 3 -bevacizumab at a dose of 10 μg/0.74MBq/mouse. For pharmacokinetics study (n = 5), five microliters of blood from the tail vein were collected at serial time points. For a biodistribution study (n = 15), 5 mice per group were anesthetized, then killed at 2 hours, 5 hours and 12 hours post injection. Tissues from blood, liver, kidney, lung, spleen, small intestine, bone, aorta artery, stomach, colon, heart, thyroid, and muscle were harvested. Each sample was weighed and counts were recorded with a counter (CRC-15R, Capintec Inc., Ramsey, NJ). After corrected for radioactive decay, data were calculated as a percent of injected dose per gram of tissue (ID%/g). The mean blood half-life of 99m Tc-MAG 3 -bevacizumab was analyzed by monoexponential decay using GraphPad Prism 5.01 (GraphPad Software Inc.).

Immunoreactivity of 99m Tc-MAG 3 -bevacizumab.
The immunoreactivity assay was conducted according to previous reported 43,44 . Each well in ELISA plates (Corning Incorporated, USA) was coated with 5 μg/ml of recombinant human VEGF165 (R&D Systems) at 4 °C overnight, and then VEGF165 solution was adjusted to pH 9.6 by adding 15 mM Na 2 CO 3 and 35 mM NaHCO 3 . Thereafter, the wells were blocked with 100 μL 1% human serum albumin (HSA, Sigma) in 0.067 M PBS. After blocking, the wells were washed three times with 0.1% polysorbate 80 (Sigma) in 0.067 M PBS. For the following binding assay, 10 ng/ml of 99m Tc-MAG 3 -bevacizumab was added to the wells with VEGF coated. All wells were allowed to incubate for 2 h at room temperature. Subsequently, unbound antibody was removed by washing the wells three times with 0.1% polysorbate 80 in 0.067 M PBS. Finally, the bound antibody was solubilized with 0.2 M NaOH and withdrawn for gamma counting. The percentage of binding was calculated as bound counts divided by total counts. Competition experiments were performed by adding an excess of unlabeled bevacizumab (1000 fold) to the wells one hour in advance before 99m Tc-MAG 3 -bevacizumab was added into VEGF165 coated wells. Each group was conducted in triplicate.
Animal protocol. Wild-type C57BL/6 J (n = 28) and ApoE −/− mice (n = 20) (male, 8weeks) were purchased from Cavens Laboratory Animal Center (Changzhou, China). C57BL/6 J mice with the age of 8 weeks were used for pharmacokinetics study (n = 5) and biodistribution study (n = 15). The rest C57BL/6 J mice (n = 8) were fed standard chow containing 4% to 6% fat and a cholesterol content <0.02% for 20 weeks and served as controls for imaging and pathology, showed in Fig. 3. The ApoE −/− mice (n = 10) fed with a Western diet (containing 21% fat and 0.15% cholesterol by weight) with a high fat/high cholesterol (HFC) diet for 20 weeks without atorvastatin therapy were used for imaging and pathology, displayed in Fig. 3. The ApoE −/− mice as treatment group (n = 10) were given 12 weeks of atorvastatin therapy with 10 mg/kg/d after 8 weeks on a Western diet applied for imaging and pathology (Fig. 3). All mice were kept in a temperature-controlled environment with a 12-hour light/dark cycle with free access to food and water. Animal care and experimental procedures were conducted in compliance with the Helsinki Declaration, and were approved by an institutional animal care committee.

The basic information of mice model and its serological biochemical analysis. All mice, both
with and without imaging, fasted for 4 hours. Body weight and the weight of abdominal fat were measured after general anesthesia by intraperitoneal injection of 4% chloral hydrate (400 mg/kg). In addition, blood samples were collected by left-ventricle puncture. After coagulating for 20 min at room temperature, the blood was centrifuged (3000 rpm × 10 min) to extract supernatant serum. Plasma TG, TC, HDL, and LDL levels were then measured enzymatically with commercial kits. Enzyme-linked immunosorbent assay (Elisa) kits were used to test serological TNF-1α, TGF-β, IL-1β and CRP. Assays were performed according to manufacturer's protocols, using a microplate reader for Elisa (Denley Dragon Wellscan MK 3, Thermo, Finland).
Micro-SPECT/CT data were transferred to software InVivo Scope (Version 1.43, Bioscan, Washington DC, USA) for processing. For each small-animal SPECT scan, 3-dimensional regions of interest (ROI) were drawn in the aortic arch, thoracic aorta and abdominal aorta in areas of obvious radioactivity (Fig. 6A). Another ROI of corresponding size was drawn in an area of surrounding waist muscle in comparison as background activity. The concentration of radioactivity (mCi/mm 3 ) was automatically generated by the software for each ROI. 99m Tc-MAG 3 -bevacizumab uptake in each section of aortic plaque was expressed as a ratio for each ROI of plaque signal to background signal (P/B), as the semi-quantitative analysis index referenced in our previous research method 45 .
Ex vivo BSGI imaging and semi-quantitative analysis. After micro-SPECT/CT imaging, the aortas were immediately removed for ex-vivo planar imaging by BSGI to eliminate adjacent tissue interference and to further verify signal intensity among different mice. BSGI was performed with the following parameters: collimator, low-energy general-purpose; peak energy, 140 keV; window width, 10%; resolution, 0.32 mm/pixel; matrix, 80 × 80. The collection time was 30 min with total counts of 80000-100000. The semi-quantitative analysis of 99m Tc-MAG 3 -bevacizumab uptake in BSGI of aortic plaque was expressed as the value of plaque to non-plaque counts ratio (P/N) 46 .
Oil-Red-O staining of aorta and analysis. After blood was drawn from each mouse, the circulation system was washed with saline, and then fixed with a 4% paraformaldehyde twice. The aorta was excised after the removal of aortic peripheral adipose tissue, and then was cut longitudinally to expose the intimal surface. At this time, samples were fixed in 4% paraformaldehyde for 24 hours. Two aortas of ApoE −/− mice, treatment mice and C57BL/6 J mice that experienced in-vivo SPECT/CT imaging and BSGI were selected for Oil-Red-O staining, as were two aortas of ApoE −/− and treatment mice and one control mouse without imaging (Fig. 3). The staining method is as follows: the fixed aortas were rinsed with 60% propylene glycol for 5 min and stained in 0.5% Oil-Red-O solution (Sigma) for 45 min. The aortas were then differentiated in a 60% propylene glycol solution for 5 min. The stained aortas were spread on a black charpie for photographing using a digital camera under identical light conditions with same the photographing parameters. Pictures were analyzed with Image Pro Plus (Version 6.0, Media Cybernetics, Washington DC, USA).
Histological Evaluation. Two aortas of ApoE −/− mice, treatment mice and C57BL/6 J mice that underwent in-vivo SPECT/CT imaging and BSGI were selected for histological evaluation. We also selected four aortas of ApoE −/− mice, treatment mice and three control mice without imaging for the same evaluation (Fig. 3). Briefly, all aortas were fixed in formalin, dehydrated, embedded in paraffin and sliced in the region of the aortic arch, thoracic aorta and abdominal aorta that presented with obvious plaque. Serial sections (5 μm thick) were prepared for Hematoxylin-Eosin (HE) and Masson trichrome staining.
Additionally, CD31 and VEGF immunohistochemical staining were also performed on the consecutively cut specimens as follows: serial sections (5 μm thick) were deparaffinized and rehydrated through a series of xylenes and graded alcohols before undergoing antigen retrieval pretreatment (sodium citrate-hydrochloric acid buffer solution for 30 min). Samples were treated with 0.3% H 2 O 2 for 30 min, followed by blocking with Fatal Bovine serum (Gibco, USA) for 1 hour to prevent nonspecific binding. The sections were then incubated overnight at 4 °C with a primary antibody (anti-CD31 antibody or anti-VEGF antibody, 1:100 in blocking serum; Abcam). A secondary antibody was then applied in GTVision TM III Detection System/Mo&Rb (Gene Tech Company Limited, shanghai, China). Digital images of the stained sections were obtained with a scanning light microscope (Leica Microsystems; Germany). Statistical analysis. All statistical analyses were performed with SPSS19.0 (IBM, Chicago, IL, USA) and GraphPad Prism 5.01 (GraphPad Software Inc.) software. Results were expressed as mean ± SD. Unpaired Student's t tests were performed to evaluate the significance of differences in body weight, the weight of abdominal fat, plasma lipid and inflammatory factor levels, as well as plaque uptake of 99m Tc-MAG 3 -bevacizumab among different groups. A two-tailed with a P-value < 0.05 was considered statistically significance.