| BackgroundAtherosclerosis(AS)is a kind of chronic progressive inflammatory diseases mainly occurs in large and medium-sized arteries,which has been proved to be the pathological basis of cardiovascular disease.Ischemia and necrosis will occur in the corresponding organs when AS plaque ruptures,thrombosis and lumen obstruction reaches a certain level,which will lead to a series of serious complications.Coronary atherosclerosis obstruction could lead to myocardial ischemic infarction,cardiac arrhythmia and even sudden death,while,cerebrovascular obstruction could cause cerebral infarction and brain atrophy,etc.At present,the examination skill of AS in clinic mainly include Arteriography,Doppler Ultrasound,Magnetic Resonance Imaging,etc.Coronary Computed Tomography Angiography(CCTA)can assess the degree of arterial lumen stenosis and analyze the size of atherosclerotic plaques.However,CCTA image acquisition requires intravenous contrast,to which some patients are allergic.Also,the instrumentation requires extremely high spatial,temporal resolution,and also high demands on the patient’s heart rate.Intravascular ultrasonography can evaluate plaque size and distribution,but the reflection of ultrasound by plaque calcification made ultrasound unable to accurately calculate calcification area and volume.Magnetic resonance imaging can assess the lumen and wall morphology in atherosclerotic arteries,but artifacts caused by wall motion are difficult to avoid.In conclusion,all of the above examinations are well for detecting larger plaques in advanced stages,and poor for examining milder AS plaques in early stages.Molecular imaging in nuclear medicine is a new technique with radionuclide labeled targeting molecular probes.Molecular imaging can reflect the physio-pathological changes of the body at the cellular and molecular levels in vivo,with high specificity and sensitivity,so it could detect lesions and make early diagnose before the anatomical structure changes.Therefore,searching for specific molecular probes to do radionuclide imaging is of great significance for the diagnosis of AS plaques with a good prospect for clinical applications.CD93 is a type I transmembrane glycoprotein,mainly expressed on the surface of macrophages(mΦ)and endothelial cells,plays an important role in the regulation of inflammation;CD93 is involved in the regulation of innate immunity,differentiation of monocyte to mΦ,and cell adhesion and phagocytosis;CD93 also promotes β1-integrin activation and fibronectin production,enhances the angiogenesis.MΦ plays a key role in the development of AS and is involved in whole process of AS progression.Within AS plaque lesions,MΦ migrates,adheres and infiltrates into the subendothelial cells,recognizes and phagocytizes oxidative-modified low-density lipoprotein(ox-LDL),then differentiates into foam cells.Foam cells programmes death and necrosis,further causing more MΦ phagocytosis and further plaque aggravation.In the development and progression of AS,inflammation plays an essential role,therefore,CD93 might be used as a new imaging target for AS.In this study,firstly,we established an AS model with SD rats,prepared 125Ⅰ-anti-CD93 mAb probe,performed phosphor autoradiography and biodistribution studies,to explore the targeting of 125Ⅰ-anti-CD93 mAb probe on AS plaque;Secondly,we prepared 3H-2-DG-CD93hi MΦ probe to explore the chemiotaxis of CD93hi MΦ on atherosclerotic plaque;The third,we prepared a CD93hi MΦ dynamic adoptive transferred ApoE-/-mouse AS model,studied the effect of CD93hi MΦ adoptive transferred on plaque severity;Finally,we preliminarily explored the underlying molecular mechanism.This study provided a new molecular target for atherosclerotic plaque molecular imaging,supplied a new strategy for dynamic monitoring of plaque tissue CD93 high expressing macrophages,and more importantly,supplied an experimental basis for targeting CD93 high expressing mΦwithin atherosclerotic plaque tissue for AS diagnosis and treatment.Part One The Preparation of 125Ⅰ-anti-CD93 mAb and Targeting to Atherosclerosis PlaqueMethods1.Preparation of AS model in SD rats:The AS model was established using SD rats with left carotid artery clamped and high-fat diet.The model was identified by H&E staining of left carotid artery,right carotid artery and aorta and Oil Red O staining of aorta.2.Preparation of 125Ⅰ-anti-CD93 mAb probe:Iodogen method was used to prepare 125Ⅰ-anti-CD93 mAb probe,with isotype 125Ⅰ-IgG as control.The labeling rate,radiochemical purity and stability were measured;SD rat peritoneal MΦ was isolated and in vitro saturation assay&competition assay were performed to explore the affinity and specificity of the probe for CD93 on mΦ.3.Phosphor autoradiography:125Ⅰ-anti-CD93 mAb or 125Ⅰ-IgG probe was injected into the SD rats model through the tail vein,and whole-body dynamic phosphor autoradiography was carried out at 24 and 48 hours,respectively.OptiQuantTM software was used for semi-quantitative analysis(DLU/mm2).At 48 hours,the bilateral carotid arteries and aorta were stripped,and ex vivo arterial phosphor autoradiography was performed.4.Biodistribution study:Rats were executed at 48 hours after tracer injection for biodistribution studies,and the bilateral carotid arteries and aorta were stripped,the major tissues were separated,and radioactivity was measured after weighing.%ID/g was calculated.Results1.H&E staining showed that atherosclerotic plaques were formed in the clamped left carotid artery and aorta of SD rats,and Oil Red O staining showed obvious lipid droplet deposition in the aorta,which confirmed that the AS model in SD rats was successfully established.2.125Ⅰ-anti-CD93 mAb/125Ⅰ-IgG probe were successfully prepared.125Ⅰ-anti-CD93 mAb bound to CD93hi MΦ with a Bmax value of 910.3 cpm/106 cells and a Kd value of 14.57 nM,and to CD93lo MΦ with a Bmax value of 385 cpm/106 cells and a Kd value of 12.43 nM.The results of saturation assay showed that the binding sites in CD93hi MΦ was significantly increased.Competitive binding assay showed that an excess of unlabeled anti-CD93 mAb could block the binding of 125Ⅰ-anti-CD93 mAb to CD93hi MΦ.3.The whole-body dynamic phosphor autoradiography showed an obvious 125Ⅰ-anti-CD93 mAb probe radioactivity accumulation in the clamped left carotid artery of SD rats,and the ex vivo arterial phosphor autoradiography demonstrated the radioactivity in the left carotid artery significantly higher than in the right carotid artery.125Ⅰ-IgG probe injection group did not show significant radioactivity accumulation in phosphor autoradiography both in vivo and in vitro(p<0.05).4.The biodistribution results showed that the radioactivity accumulation of 125Ⅰ-anti-CD93 mAb probe in the clamp left carotid artery was significantly higher than in the right carotid artery,and the 125Ⅰ-anti-CD93 mAb probe radioactivity accumulation in the left carotid artery was significantly higher than 125Ⅰ-IgG probe(T/NT ratio:2.58±0.13,0.64± 0.03,p<0.01).Part Two The Chemotaxis of 3H-2-DG-CD93hi MΦ to Atherosclerosis Plaque in vivoMethods1.Preparation of CD93hi/lo MΦ:SD rat peritoneal MΦ was isolated,LPS was used to prepare CD93hi MΦ(0.1μg/ml,12h),RT-PCR,Western blot were performed to identify the expression of CD93.CD93lo MΦ was prepared without LPS stimulation.2.3H-2-DG incorporation:3H-2-DG(4.0 MBq)was incorporated into CD93hi/lo MΦ(1.5×107/each),the effects on MΦ morphology and phagocytosis of Dil-ox-LDL function were observed under microscopy,and the effects on cell cycle and cell apoptosis were detected by Flow cytometry.3.Phosphor autoradiography and biodistribution:3H-2-DG-CD93hi/lo MΦ was injected intraperitoneally into the SD rat AS model(3.7 MBq/each rat),and whole-body dynamic tritium screen autoradiography was performed at 24,48 and 72 hours,ex vivo aorta tritium screen autoradiography and biodistribution studies were performed at 72 hours.4.Immunofluorescence and Immunohistochemical staining:Immunofluorescence and immunohistochemical staining of CD93 and CD68 in isolated carotid arteries and aorta were performed.Results1.RT-PCR and Western blot showed that CD93 expression was significantly elevated at the mRNA level and protein level after LPS stimulation(p<0.05).2.3H-2-DG-CD93hi/lo MΦ was successfully prepared,and 3H-2-DG incorporation had no significant effect on MΦ morphology,phagocytosis of Dil-Ox-LDL,cell apoptosis,and cell cycle.3.Ex vivo aorta autoradiography showed the radioactivity accumulation of 3H-2-DG-CD93hi MΦ probe was stronger than 3H-2-DG-CD93lo MΦ probe.4.The biodistribution results showed that radioactivity accumulation of the 3H-2-DG-CD93h’MΦ probe in the clamped left carotid artery and aorta was increased with time going;at 72 hours,the radioactivity accumulation of the 3H-2-DG-CD93hi MΦ probe in the clamped left carotid artery was significantly higher than that of the 3H-2-DG-CD93lo MΦ(p<0.001)and 3H-2-DG(p<0.05),which indicated that the chemotaxis of CD93hi MΦ to AS plaque was higher and accumulated at the plaque specifically.5.Immunofluorescence staining of isolated arteries showed co-expression of CD93 and CD68 at the plaque,and immunohistochemical results showed higher expression of CD93 and CD68 in the left carotid artery than in the right carotid artery.Part Three CD93hi MΦ Adoptive Transfer Promoted the Development of Atherosclerosis PlaqueMethods1.Establishment of ApoE-/-mice AS model:ApoE-/-mice was used to establish AS model with left carotid artery clamped and high-fat diet.The Models were divided into three groups,Non-MΦ(no MΦ adoptive transferred)adoptive transferred group,CD93hi MΦ adoptive transferred group,and CD93lo MΦ adoptive transferred group.Among ApoE-/-mice model groups with CD93hi/lo MΦ adoptive transferred,CD93hi/lo MΦ was dynamically adoptive transferred at week 1,4,8,12 and 16 of modeling(5×106/each mouse),respectively.At week 12 and 20,H&E staining.Oil Red O staining and serum lipid measurement were performed for the bilateral carotid arteries and aorta to evaluate the severity of AS plaques.2.Fluorescence imaging:At week 20,GFP(green fluorescence protein)labeled CD93hi/lo MΦ(from C57BL/6-Tg-Osb/J mouse)were adoptive transferred into ApoE-/-mouse model(5×106/each mouse),and the bilateral carotid arteries and aorta were stripped for fluorescence imaging after 72 hours to investigate the chemotaxis of CD93hi/lo MΦ to AS plaque.Non-AS model C57BL/6 mouse was used as control.3.Immunohistochemical staining:Immunohistochemical staining for CD93,F4/80,ApoE,and CD31 was performed on the bilateral carotid arteries and aorta in CD93hi/lo MΦ adoptive transferred ApoE-/-mice models at week 12 and 20.4.Phosphor-autoradiography and biodistribution studies:125Ⅰ-anti-CD93 mAb probe was prepared and injected into mice by tail vein for phosphor-autoradiography and biodistribution studies in CD93hi/lo MΦ adoptive transferred ApoE-/-mice models at week 12,and in the above three groups at week 20 to explore the targeting of CD93 probes for AS plaques and plaque severity in the CD93hi/lo MΦ adoptive transferred ApoE-/-mice models.Results1.Fluorescence imaging results showed a stronger fluorescence signal at the arterial plaque after 72 hours of CD93hi MΦ injection,compared with the group with CD9310 MΦ injection in AS models,which indicated that CD93hi MΦ had a stronger chemotaxis to the AS plaque.While,no fluorescence signal was found in non-AS model C57BL/6 mice.2.H&E and Oil Red O staining showed more severe AS plaques were performed in CD93hi MΦ adoptive transferred ApoE-/-mouse model,compared with CD93lo MΦ and Non-MΦadoptive transferred ApoE-/-mouse model at week 20,while no significant difference between them at week 12.The results of blood lipid tests showed that TC and LDL levels in CD93hi/lo MΦ adoptive transferred groups were significantly increased at week 20,and the level of CD93hi MΦ adoptive transferred ApoE-/-mice was higher than that of CD93lo MΦ.3.Immunohistochemical staining results showed the expression of CD93,F4/80,CD31 was significantly increased in the arteries of CD93hi MΦ adoptive transferred ApoE-/-mouse model,compared with CD93lo MΦ and Non-MΦ adoptive transferred ApoE-/-mouse model at week 20.4.Ex vivo aorta phosphor-autoradiography and biodistribution results showed obvious radioactivity accumulation in AS plaque in Non-MΦ adoptive transferred ApoE-/-mouse model at week 20,CD93 probe showed a good targeting for AS plaque.Stronger radioactivity accumulation was shown in ex vivo aorta phosphor autoradiography in CD93hi MΦ adoptive transferred ApoE-/-mouse model.Radioactivity accumulation of 125Ⅰ-anti-CD93 mAb probe group in the left carotid artery in CD93hi MΦ adoptive transferred ApoE-/-mouse model was significantly higher than in CD93lo MΦ adoptive transferred ApoE-/-mouse model,which confirmed that CD93hi MΦ dynamic adoptive transferred promoted the progression of AS plaque.Part Four The Mechanisms of CD93hi MΦ on the Development of Atherosclerosis Methods1.AS-related protein expression in artery:Protein microarray was used to analyze the expression of inflammation-related molecules in the left carotid artery of CD93hi/lo MΦadoptive transferred ApoE-/-mice.2.Cytokines secretion:ELISA was used to measure IL-1β and TNF-α levels in CD93hi/lo MΦculture supernatants and in the serum of CD93hi/lo MΦ adoptive transferred ApoE-/-mice at w20.3.Western blot:The expression of CD93,p-STAT3,STAT3,p-NF-κB and NF-κB on CD93hi/lo MΦ were performed.4.Immunohistochemistry staining:The expression of STAT3 on aorta in CD93bi/lo MΦadoptive transferred ApoE-/-mice was performed.Results1.Protein microarray results showed that the expression of pro-inflammatory factors,chemokines and proangiogenic factors was significantly higher,and immunosupressed factor lower in the left carotid artery of CD93hi MΦ ApoE-/-mice than in CD93lo MΦ ApoE-/-mice.2.The level of IL-1β and TNF-α in CD93hi MΦ culture supernatant was significantly higher than that in CD9310 MΦ culture supernatant(p<0.05);the level of serum IL-1β and TNF-α in CD93hi MΦ adoptive transferred ApoE-/-mice was significantly higher than that in CD93lo MΦ adoptive transferred ApoE-/-mice(p<0.05).3.The expression of p-STAT3,p-NF-κB in CD93hi MΦ was higher than CD93lo MΦ(p<0.05).Immunohistochemistry staining confirmed the expression of STAT3 on aorta.Conclusions1.Successfully prepared 125Ⅰ-anti-CD93 mAb molecular probe,which could specifically bind to CD93hi MΦ.Phosphor-autoradiography and biodistribution studies showed that this probe could highly accumulate at the plaque after injection,CD93 may be used as a specific target for imaging AS plaque.2.The 3H-2-DG-CD93hi MΦ probe was successfully prepared,tritium screen phosphor-autoradiography and biodistribution study confirmed this kind macrophage exhibited a high chemotaxis to AS plaques.3.In ApoE-/-mice AS model,CD93hi/lo MΦ was dynamically adoptive transferred at week1,4,8,12,16 and pathological analysis,serum lipid levels,as well as the expression of inflammatory molecules associated with diseased vessels demonstrated that CD93hi MΦadoptive transferred promoted the progression of AS plaques.4.CD93hi MΦ promotes AS plaque progression mainly through the p-STAT3/p-NF-κB to enhance the pro-inflammatory factor secretion and neovascularization..Innovation1.Successfully prepared a new molecular probe 125Ⅰ-anti-CD93 mAb,which showed a good targeting to AS plaque,CD93 in plaque may be as a new molecular imaging target for AS.2.MΦ with CD93 high expression exhibited a high chemotaxis to atherosclerotic plaque,which may be as a new target for atherosclerotic vulnerable plaque monitoring.3.CD93hi MΦ promoted AS progression mainly through the p-STAT3/p-NF-κB to enhance the pro-inflammatory factor secretion and neovascularization. |
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