靶向LOX-1的ApoE-/-小鼠动脉粥样硬化斑块近红外荧光成像及阿托伐他汀干预实验研究
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摘要
目的探讨靶向LOX-1的近红外分子探针示踪小鼠动脉粥样硬化(AS)斑块及评价阿托伐他汀干预的可行性。材料与方法采用12周龄Apo E-/-小鼠经高脂高糖饮食喂养16周建立AS模型,C57小鼠作为对照。基线实验分为靶向探针组(n=5)、竞争性抑制组(n=4)、碱性磷酸盐溶液(PBS)组(n=5)及对照C57组(n=5),经小鼠尾静脉注入1 mg/kg靶向探针或PBS,24 h后行近红外荧光成像(NIRF)。干预实验将Apo E-/-小鼠随机分为对照组和干预组(n=5,饮食中不加或加阿托伐他汀干预6周)。采用油红O染色鉴定斑块面积。斑块面积与NIRF信号分布之间的关系采用线性相关分析。结果 Apo E-/-小鼠注入anti-LOX-1-NIR797探针24 h后,NIRF示主动脉斑块区见强荧光信号,竞争性抑制组斑块SNR显著降低,而对照组C57小鼠注入靶向探针或Apo E-/-小鼠注入PBS时,其主动脉斑块信噪比较弱(P<0.01)。实验组Apo E-/-小鼠主动脉油红O染色和NIRF的阳性面积呈正相关(r=0.917,P<0.001)。阿托伐他汀干预组anti-LOX-1-NIR797信号较对照组显著降低、腹主动脉斑块面积减少(P<0.05)。结论应用靶向LOX-1的近红外探针在NIRF平台可有效检测Apo E-/-小鼠AS斑块并评价阿托伐他汀的干预效果。
Purpose To explore the feasibility of detecting the progression and regression of atherosclerotic plaques with near-infrared fluorescence imaging(NIRF) using LOX-1 targeted imaging probes. Materials and Methods Atherosclerotic plaques were established in male atherosclerotic Apo E-/-mice by feeding a high-glucose-high-fat diet for 16 weeks starting at age of 12 weeks. Correspondingly C57 BL/6 mice were used as negative controls. In the baseline experiments, anti-LOX-1-NIR797 was injected into 9 Apo E-/-mice via tail vein, while 4 Apo E-/-mice received unlabeled antibodies before tracer injection as the competitive inhibition group and another 5 received PBS injection as the PBS control group. Correspondingly, 5 wild-type C57 mice fed normal chow received the probe. NIRF was performed 24 hours after tail vein injection of the probe. For the intervention experiment, 10 mice were randomly assigned into control group and intervention group then were given another 6 weeks of an HCD spiked with(regression group) or without(progression group) atorvastatin. The plaque areas were investigated using Oil Red O(ORO) staining. The correlation between the plaque area and antibody uptake as assessed by NIRF was performed using linear correlation analysis. Results In Apo E-/-mice, a significant fluorochrome accumulation in atherosclerotic plaques was demonstrated in aortic arch, bifurcations of aorta and abdominal aorta 24 hours after injection of anti-LOX-1-NIR797 probe. Minimal antibody uptake was observed in normal vessels from C57 mice receiving the anti-LOX-1-NIR797 and in atherosclerotic vessels from Apo E-/-mice receiving the competitive LOX-1-m Ab or PBS, which were different from the uptake of LOX-1 targeted group(P<0.01). There was a linear correlation in positive area between NIRF imaging and ORO staining in the aortas(r=0.917, P<0.001, n=8). Furthermore, compared with control group, the atherosclerotic lesions of atorvastatin-treated mice showed reduced antiLOX-1-NIR797 uptake and plaque areas in abdominal aorta(P<0.05). Conclusion This study indicates that NIRF plaque imaging is feasible with a LOX-1-targeted NIRF probe in Apo E-/-mice. Thus, LOX-1-based molecular imaging of atherosclerotic plaques is feasible and may provide effective methods for monitoring the response to therapeutic interventions for atherosclerosis.
Purpose To explore the feasibility of detecting the progression and regression of atherosclerotic plaques with near-infrared fluorescence imaging(NIRF) using LOX-1 targeted imaging probes. Materials and Methods Atherosclerotic plaques were established in male atherosclerotic Apo E-/-mice by feeding a high-glucose-high-fat diet for 16 weeks starting at age of 12 weeks. Correspondingly C57 BL/6 mice were used as negative controls. In the baseline experiments, anti-LOX-1-NIR797 was injected into 9 Apo E-/-mice via tail vein, while 4 Apo E-/-mice received unlabeled antibodies before tracer injection as the competitive inhibition group and another 5 received PBS injection as the PBS control group. Correspondingly, 5 wild-type C57 mice fed normal chow received the probe. NIRF was performed 24 hours after tail vein injection of the probe. For the intervention experiment, 10 mice were randomly assigned into control group and intervention group then were given another 6 weeks of an HCD spiked with(regression group) or without(progression group) atorvastatin. The plaque areas were investigated using Oil Red O(ORO) staining. The correlation between the plaque area and antibody uptake as assessed by NIRF was performed using linear correlation analysis. Results In Apo E-/-mice, a significant fluorochrome accumulation in atherosclerotic plaques was demonstrated in aortic arch, bifurcations of aorta and abdominal aorta 24 hours after injection of anti-LOX-1-NIR797 probe. Minimal antibody uptake was observed in normal vessels from C57 mice receiving the anti-LOX-1-NIR797 and in atherosclerotic vessels from Apo E-/-mice receiving the competitive LOX-1-m Ab or PBS, which were different from the uptake of LOX-1 targeted group(P<0.01). There was a linear correlation in positive area between NIRF imaging and ORO staining in the aortas(r=0.917, P<0.001, n=8). Furthermore, compared with control group, the atherosclerotic lesions of atorvastatin-treated mice showed reduced antiLOX-1-NIR797 uptake and plaque areas in abdominal aorta(P<0.05). Conclusion This study indicates that NIRF plaque imaging is feasible with a LOX-1-targeted NIRF probe in Apo E-/-mice. Thus, LOX-1-based molecular imaging of atherosclerotic plaques is feasible and may provide effective methods for monitoring the response to therapeutic interventions for atherosclerosis.
引文
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[13]Prati F,Regar E,Mintz GS,et al.Expert review document on methodology,terminology,and clinical applications of optical coherence tomography:physical principles,methodology of image acquisition,and clinical application for assessment of coronary arteries and atherosclerosis.Eur Heart J,2010,31(4):401-415.
[14]Briley-Saebo KC,Cho YS,Shaw PX,et al.Targeted iron oxide particles for in vivo magnetic resonance detection of atherosclerotic lesions with antibodies directed to oxidationspecific epitopes.J Am Coll Cardiol,2011,57(3):337-347.
[15]Nahrendorf M,Keliher E,Marinelli B,et al.Hybrid PEToptical imaging using targeted probes.Proc Natl Acad Sci USA,2010,107(17):7910-7915.
[2]Libby P,Ridker PM,Hansson GK.Progress and challenges in translating the biology of atherosclerosis.Nature,2011,473(7347):317-325.
[3]Hansson GK,Libby P,Tabas I.Inflammation and plaque vulnerability.J Intern Med,2015,278(5):483-493.
[4]Goyal T,Mitra S,Khaidakov M,et al.Current concepts of the role of oxidized LDL receptors in atherosclerosis.Curr Atheroscler Rep,2012,14(2):150-159.
[5]Pothineni NVK,Karathanasis SK,Ding Z,et al.LOX-1 in atherosclerosis and myocardial ischemia:biology,genetics,and modulation.J Am Coll Cardiol,2017,69(22):2759-2768.
[6]Wen S,Liu DF,Cui Y,et al.In vivo MRI detection of carotid atherosclerotic lesions and kidney inflammation in Apo Edeficient mice by using LOX-1 targeted Iron nanoparticles.Nanomedicine,2014,10(3):639-649.
[7]Lu T,Wen S,Cui Y,et al.Near-infrared fluorescence imaging of murine atherosclerosis using an oxidized low density lipoprotein-targeted fluorochrome.Int J Cardiovasc Imaging,2014,30(1):221-231.
[8]Wen S,Liu DF,Liu Z,et al.Ox LDL-targeted Iron oxide nanoparticles for in vivo MRI detection of perivascular carotid collar induced atherosclerotic lesions in Apo Edeficient mice.J Lipid Res,2012,53(5):829-838.
[9]卢瞳,文颂,周官辉,等.动脉粥样硬化斑块MRI和近红外分子影像的实验研究.中华核医学与分子影像杂志,2012,32(1):16-21.
[10]Fuster V,Moreno PR,Fayad ZA,et al.Atherothrombosis and high-risk plaque:part I:evolving concepts.J Am Coll Cardiol,2005,46(6):937-954.
[11]周长武,赵锡海,李澄.胸主动脉粥样硬化斑块影像学研究进展.中国医学影像学杂志,2016,24(5):384-387,391.
[12]Vinegoni C,Botnaru I,Aikawa E,et al.Indocyanine green enables near-infrared fluorescence imaging of lipid-rich,inflamed atherosclerotic plaques.Sci Transl Med,2011,3(84):84ra45.
[13]Prati F,Regar E,Mintz GS,et al.Expert review document on methodology,terminology,and clinical applications of optical coherence tomography:physical principles,methodology of image acquisition,and clinical application for assessment of coronary arteries and atherosclerosis.Eur Heart J,2010,31(4):401-415.
[14]Briley-Saebo KC,Cho YS,Shaw PX,et al.Targeted iron oxide particles for in vivo magnetic resonance detection of atherosclerotic lesions with antibodies directed to oxidationspecific epitopes.J Am Coll Cardiol,2011,57(3):337-347.
[15]Nahrendorf M,Keliher E,Marinelli B,et al.Hybrid PEToptical imaging using targeted probes.Proc Natl Acad Sci USA,2010,107(17):7910-7915.