The Effects Of Kallistatin On Atherosclerosis And Development Of Melanin Nanoparticles Targeting Macrophages In Atherosclerosis

?.Molecular mechanisms underlying the antiatherosclerotic effects of kalliststinObject:To investigate the effect of kallistatin?KS?on atherosclerosis inflammation and its possible mechanism.Methods:We established the atherosclerosis model in apoE^-/-mice via partial left carotid artery?PLCA?ligation.The human kallistatin gene in an adenoviral vector?Ad.HKS?was delivered via tail vein before PLCA ligation.The mice were divided into 2groups:PLCA+Ad.HKS and PLCA+adenoviral vector?Ad.Null?for two and four weeks respectively.MRI and pathological staining were used to detect the progress of atherosclerosis.Real-time PCR was used to detect the polarization of macrophages.Futhermore,RAW 264.7 macrophages cells were treated with the human kallistatin?HKS?and Krüppel-like factor 4?KLF4?small interfering RNA oligonucleotide?KLF4siRNA?.The polarization of macrophages was determined by Flow cytometry.Western blot was applied to find the effect of KLF4 associated with M1/M2 biomaker.Results:Human kallistatin was expressed in the mice after kallistatin gene delivery.And kallistatin significantly inhibits plaque formation and reduces inflammation in plaque and liver four weeks after gene delivery.Moreover,M1 macrophage markers inducible nitric oxide synthase?iNOS?,monocyte chemotactic protein-1?MCP-1?expression decreased and M2 macrophage markers arginase 1?Arg1?,interleukin 10?IL-10?expression increased after gene delivery.Furthermore,in cultured RAW 264.7macrophages cells,Kallistatin significantly stimulated M2 makers expression and population,decreased M1 marker expression by real time PCR,and these effects were blocked by KLF4 siRNA.Conclusions:These findings demonstrated that kallistatin inhibits atherosclerosis plaque formation and regulates M1/M2 macrophages polarization via KLF4 activation.?.Evaluation of ?v?3 integrin-targeted PET tracer 89Zr-RGDMNP for imaging of atherosclerotic plaque in Apo E-/-miceObjective: This study evaluate that 89Zr-RGD-MNP is a PET tracer binding specifically to ???3.Therefore,89Zr-RGD-MNP PET imaging of ???3 expression in mice carotid plaques might provide a novel noninvasive biomarker of atherosclerotic plaque.Methods: We conjugated ???3 integrins,cyclic c?RGDf C?peptide,to ultrasmall melanin nanoparticle?MNP?and incorporated the long-lived positron-emitting nuclide 89 Zr to allow for atherosclerotic plaque accumulation.The surface morphology and structure was detected by Transmission Electron Microscope?TEM?,the size was detected by Dynamic Light Scattering?DLS?,the cytotoxicity was detected by MTT and the targeting ability was detected by Fontana Masson silver staining.Apo E-/-mice were fed a high-fat diet for more than 2 weeks.Branches of the left carotid artery in apo E-/-mice were ligated to produce the partial left carotid artery?PLCA?model.14 days after surgery,they were injected with 89Zr-RGD-MNP followed by imaging with a small animal PET scanner at different time points.The carotid artery and the major organs was collected after PET imaging for biodistribution studies.Results:The melanin nanometer probe 89Zr-RGD-MNP were prepared successfully.The nanoparticles exhibited a good spherical morphology,the mean size was less than 10 nm and with narrow diameter distribution.MTT assay showed no significant effect on cell activity,and Fotana-Masson silver staining showed high targeting to macrophages.PET imaging showed specific tracer accumulation at plaques in the left carotid artery,confirmed by competitive receptor blocking studies and the contrast in the right carotid atery.In the biodistribution studies,the left carotid?5.29%±0.78%?showed higher uptake than the right carotid?2.11%±1.55%?.Conclusions: We have developed 89Zr-labeled atherosclerotic plaques imaging agents based on the melanin nanoparticle.The agents have been successfully prepared with a stable physical and chemical properties,small size,good dispersibility and high biosafety.89Zr-RGD-MNP demonstrates specific tracer accumulation in mice atherosclerotic carotid plaques.In this model,its uptake was associated with ???3 expression.89Zr-RGD-MNP is a potential tracer for noninvasive imaging in atherosclerosis.