Mechanisms And Visualizations Of Myeloperoxidase Regulating Plaque Vulnerability In Atherosclerosis

Background and aim:Rupture of atherosclerotic plaques and subsequent acute thrombosis are the main causes of acute coronary syndrome.The typical vulnerable plaque has a thin fibrous cap,a lipid-rich necrotic core,abundant inflammatory cells,microcalcifications,neovessels and intraplaque hemorrhage（IPH）.However,clinicians are still faced with limited knowledge about the mechanism of vulnerable plaque rupture and challenges in accurately identifying vulnerable plaques.Myeloperoxidase（MPO）,an inflammatory protein,is secreted as an active enzyme by macrophages and neutrophils in atherosclerosis.Previous studies found that MPO may participate in plaque erosion by inducing endothelial cell apoptosis and dysfunction.The mechanism of MPO regulating plaque vulnerability is not fully understood.Therefore,this study aimed to explore the role of MPO in atherosclerotic plaque vulnerability,with a particular focus on the MPO regulating intraplaque neovessel and IPH via damaging endothelial cell.The effect of pharmacological inhibition of MPO activity on plaque vulnerability was also investigated.Furthermore,an imaging nanoprobe targeting active MPO was constructed for molecular imaging of active MPO in atherosclerosis.Additionally,we intended to use novel imaging techniques for the identification of IPH,which may help detect and monitor atherosclerotic vulnerable plaques.Methods:1.A total of 30 human carotid specimens were collected from patients undergoing carotid endarterectomy.Apo E^-/-mice were employed to establish two animal models of atherosclerotic vulnerable plaques:Apo E^-/-mice were fed with high fat diet over 40-42 weeks to establish vulnerable plaques within abdominal aorta,and Apo E^-/-mice on high fat diet underwent a tandem stenosis（TS）surgery to establish vulnerable plaques within right carotid artery.C57BL/6J mice were intramuscularly injected with Matrigel implants containing MPO and glucose oxidase to obtain an animal model of MPO embedded in Matrigel.2.Active MPO-targeting 5-HT-Fe₃O₄-Cy7 nanoprobe was synthesized by conjugating superparamagnetic Fe₃O₄@PEG-COOH with 5-HT and Cy7-NHS,and characterized using transmission electron microscopy,Malvern instrument,UV-Vis-NIR and fluorescence spectrophotometer,IVIS spectrum and magnetic particle imaging（MPI）scanner.3.MPI,fluorescence imaging（FLI）,computed tomography and magnetic resonance imaging were performed on the animals for molecular imaging of active MPO and IPH imaging.Ex vivo light sheet microscopy scanning was performed to analyze neovessel permeability and plaque size.4.Western blot,q PCR,pathological staining,immunohistochemistry and immunofluorescence staining were carried out to characterize plaque components,including MPO,macrophage,T lymphocyte,smooth muscle cell,monocyte chemoattractant protein-1,neovessel,IPH,calcification,collagen,platelet,fibrin,red blood cell,hemoglobin and ferritin.5.Cell counting kit-8 assay was performed to evaluate the cytotoxicity of 5-HT-Fe₃O₄-Cy7 nanoprobe and erythrophagocytosis assay was carried out to obtain hemosiderin for MPI and transmission electron microscopy scanning.Results:1.The role of MPO in atherosclerotic vulnerable plaque.After 40-42 weeks of high fat diet on Apo E^-/-mice,atherosclerotic plaques in the abdominal aorta exhibited more vulnerable features than did those in the aortic arch,including more macrophages,neovessels,and microcalcifications（P<0.05）.MPO was significantly higher in plaques in the abdominal aorta than in those of the aortic arch（P<0.05）.Despite no effects on plaque sizes,inhibition of MPO activity reduced macrophages,smooth muscle cells,and expression of monocyte chemoattractant protein-1 in the abdominal aorta plaques（P<0.05）.2.Synthesis and characterization of active MPO-targeting nanoprobe.Active MPO-targeting 5-HT-Fe₃O₄-Cy7 nanoprobe was mono-dispersed and stable,and had an average size of 20.97±2.23nm.In vitro imaging assays found that the concentration of 5-HT-Fe₃O₄-Cy7 nanoprobe showed a linear correlation with FLI and MPI signals.Cell and animal studies preliminarily confirmed the biological safety of 5-HT-Fe₃O₄-Cy7 nanoprobe,which was mainly distributed to liver and spleen in vivo.In vitro and in vivo targeting assays confirmed the self-oligomerization of 5-HT-Fe₃O₄-Cy7 nanoprobe in the presence of active MPO,which enabled the MPI/FLI molecular imaging of active MPO.3.MPI/FLI molecular imaging of active MPO in atherosclerosis.After intravenous injection of 5-HT-Fe₃O₄-Cy7 nanoprobe,MPI（P<0.001）and FLI signals（P<0.05）were significantly higher in plaques in the abdominal aorta than in those of the aortic arch,indicating that 5-HT-Fe₃O₄-Cy7 nanoprobe can sensitively differentiate and accurately localize vulnerable plaques via MPI/FLI molecular imaging.In addition,pharmacological inhibition of MPO activity significantly attenuated MPI and FLI signals in the abdominal aorta plaques,suggesting that 5-HT-Fe₃O₄-Cy7 nanoprobe with MPI/FLI molecular imaging enabled quantitative monitoring of MPO activity in the pharmacological intervention of atherosclerosis.4.The mechanism of MPO regulating atherosclerotic IPH.We applied a tandem stenosis to Apo E^-/-mice on high fat diet to establish the right carotid vulnerable plaques,which were characterized by IPH,intraplaque neovessels,abundant inflammatory cells,intraluminal microthrombus and necrotic core.In the human and murine carotid plaques,we observed the presence of IPH in the co-localization areas of MPO with neovessels.Despite no effects on neovessel densities or lumen areas,inhibition of MPO activity reduced endothelial v WF（P<0.01）,ameliorated neovessel permeability（P<0.05）,and decreased IPH size in carotid vulnerable plaques（P<0.0001）.5.Label-free MPI imaging of atherosclerotic IPH.The IPH in the human carotid endarterectomy samples exhibited endogenous MPI signals.In vitro studies found that the endogenous MPI signals in atheromatous plaques were attributed to superparamagnetic hemosiderin,which allows IPH detection without external MPI tracers.MPI of TS Apo E^-/-mice identified IPH in vulnerable plaques of right carotid artery,of which MPI signals were significantly higher compared with stable plaques in brachiocephalic artery（P<0.01）or normal arterial wall in left carotid artery（P<0.01）.We observed a dynamic change of IPH at 4,7 and 11 weeks post TS,and demonstrated that the IPH dynamics may be associated with neovessel permeability by in vivo MPI scanning and ex vivo light sheet microscopy scanning.Moreover,MPI identified the small-size IPH at 4 weeks post TS that MRI could not image.Conclusions:1.MPO is highly expressed in atherosclerotic vulnerable plaques.MPO contributes to plaque vulnerability by promoting IPH formation via inducing endothelial damage and increasing neovessel permeability.Inhibition of MPO activity stabilizes vulnerable plaques by reducing IPH,macrophages,smooth muscle cells and expression of monocyte chemoattractant protein-1.2.5-HT-Fe₃O₄-Cy7 nanoprobe enables MPI/FLI molecular imaging of active MPO,which may serve as a method for detecting vulnerable atherosclerotic plaques and monitoring MPO activity in atherosclerosis.3.Magnetic particle imaging is a promising technology that allows the label-free identification of atherosclerotic plaques with IPH and may help detect and monitor vulnerable plaques in patients.