| Objective To investigate whether CD137-CD137 L signal(referred to as CD137 signal)promotes the atherosclerotic calcification mediated by NFATc1 via exosomes.Methods 1.Mouse model of atherosclerosis: Apo E-/-mice fed with high diet were used to establish the model of atherosclerosis.Exosomes were tail vein injected into Apo E-/-mice.2.Transmission Electron Microscope: observe exosome expression in Apo E-/-mouse plaques and identification of isolated vesicles.3.Nanoparticles intensive analysis(NTA)and BCA method: Nanoparticle tracer analysis The diameter and concentration analysis of exosomes in each smooth muscle cell treatment group were compared,and the protein content were tested by BCA method.4.Fluorescence microscopy: Exosomes labeled by PKH-26 were cocultured with VSMCs.Then Fluorescence used microscopy to observe exosomes internalization by VSMCs 5.Western blot: Western blot were used to detect the protein expression of CD63,CD81,bone morphogenic protein-2(BMP-2),Runt-related transcription factor 2(Runx-2)and alpha smooth muscle Actin(?-SMA).6.Lentivirus infection: The coding sequence of mice sh NFATc1 was cloned into a eukaryotic expression vector PLKO.1-puro at the Eco RI/Age I site,namely PLKO.1-sh NFATc1.And PLKO.1-sh GFP was used as a control.The mouse aortic were infected the lentivirus to establish the stable cell line in the presence of polybrene.7.Masson's trichrome assay and Immunostaining: Immunostaining were used to observe the changes of NFATc1,BMP2,Runx2 in the plaque after interfering by each group's exosomes.8.Calcification detection assays: The ALP activities,von kossa staining and calcium content were used to measure the Calcification of VSMCs.The calcium content and von kossa was used to detect the vascular calcification in Apo E-/-mice.Results1.The CD137 signal influences exosome expression in Apo E-/-mice: Transmission electron microscopy showed the expression of exosomes in Apo E-/-mice plaques were significantly increased in CD137 excitation group.2.The CD137 signal influences exosome secretion in vascular smooth muscle cells: The Nanoparticle tracking analysis showed that the concentration of exosomes in CD137 excitation group were obviously higher than control group.BCA test showed the total protein level were significantly improved in CD137 excitation group compared to control group.3.CD137 signal regulates the expression of NFATc1 both in VSMCs and exosomes: The expression of NFATc1 protein increased significantly in VSMCs and exosomes of CD137 excitation group,compared to the control group(P<0.05).And the expression of NFATc1 protein level in both VSMCs and exosomes significantly increased in CD137 excitation group,compared to the control group(P<0.05).4.The influences of each group exosomes in VSMCs calcification: the expressions of BMP-2 and Runx-2 proteins in CD137 excitation group exosomes group increased significantly(P<0.01),while the expression of ?-SMA significantly decreased(P<0.01),Compared to control group.The expressions of BMP-2 and Runx-2 proteins decreased significantly in silenced NFATc1+CD137 excitation group exosomes group compared to CD137 excitation group exosomes group(P<0.01),while the expression of ?-SMA increased significantly(P<0.01).VSMCs calcification level and ALP activities in CD137 activation group exosomes group were more than those in control group exosomes group(P<0.01),silenced NFATc1+CD137 excitation exosomes group were significantly lower than those in CD137 excitation exosomes group(P<0.01).Von kossa staining showed that calcification degree of VSMCs in CD137 excitation exosomes group significantly increased(P<0.01).The calcification degree of silenced NFATc1 + CD137 excitation exosomes group were significantly decreased in CD137 excitation group exosomes group(P<0.01).5.The influences of each group exosomes in Apo E-/-mice calcification: Von kossa staining showed Apo E-/-mice aortic calcium deposits in CD137 activation group exosomes treatment group were more than those in control group exosomes group(P<0.01),silenced NFATc1+CD137 excitation group exosomes treatment group were significantly lower than those in CD137 activation group exosomes treatment group(P<0.05).Immunohistochemistry showed that expressions of BMP-2 and Runx-2 proteins in CD137 excitation group exosomes group increased significantly(P<0.01),Compared to control group.The expressions of BMP-2 and Runx-2 proteins decreased significantly in silenced NFATc1 + CD137 excitation group exosomes group compared to CD137 excitation group exosomes group(P<0.01).Conclusion CD137 signal activation promotes secretion of vascular smooth muscle cell exosomes,and may promote vascular smooth muscle cell calcification and atherosclerotic plaque calcification in Apo E-/-mice through NFATc1 by exosomes. |
PDF Full Text Request