CD137-CD137L Signaling Influences Secretion Of Vascular Smooth Muscle Cell-Derived Exosomes VIA Rab7Pathway In Mouse

ObjectiveTo investigate whether CD137-CD137 L signaling can influence the secretion of exosomes steming from mouse vascular smooth muscle cells(VSMCs)through autophagy mediated by Rab7 molecule.MethodsPrimary culture of C57BL/6J mouse thoracic aorta VSMCs was performed by tissue block adherence method.VSMCs between the third to fifth generations were isolated and cultured.VSMCs were divided into 4 groups: control group,CD137 agonist group,lentivirus control group,Rab7 lentiviral interference group.VSMCs in CD137 agonist group were treated with recombinant protein of CD137L(10 ?g/ml),VSMCs in lentivirus control group were treated with lentiviral followed by recombinant protein of CD137L(10 ?g/ml),VSMCs in Rab7 lentiviral interference group were treated with Rab7 lentiviral intervention followed by recombinant protein of CD137L(10 ?g/ml).Western blot was used to detect the protein expression of LC3?,p62,Rab7,CD9,CD81 and Hsc70.Fluorescence microscopy was used to track the changes of autophagy in cells which was infected with mRFP-GFP-LC3.Transmission electron microscope was used to observe the morphology and size of VSMC-derived exosomes.The nanoparticle tracking analysis(NTA)was used to detect the concentration and size of exosomes in each group.Results(1)Under transmission electron microscopy,The size of the vesicular structure derived from VSMC is about 30-150 nm.The exosome markers(CD9,CD81)can be detected in vesicles by western blot.(2)The expression of Rab7,LC3 ? and p62 protein in VSMC of CD137 activation group was significantly higher than that of the control group(P<0.05).The expression of Rab7,LC3 ? and p62 protein in Rab7 lentivirus interference group was lower than that in CD137 activation group(P<0.05).There was no difference between the lentivirus control group and the CD137 activation group(P>0.05).(3)The VSMC autophagic flow of each group showed that the total number of fluorescent spots and yellow fluorescent spots in the VSMC of the CD137 activation group were higher than those in the control group(P<0.05),and the number of yellow fluorescent spots in the VSMC of the CD137 activation group was more than red.The total number of fluorescent spots and yellow fluorescent spots in the VSMC of the Rab7 lentivirus interference group were less than those in the CD137 activation group(P<0.05),and the number of red fluorescent spots in VSMC of Rab7 lentivirus interference group was higher than yellow.There was no difference between the lentivirus control group and the CD137 activation group(P>0.05).(4)The secretion level of exosomes in VSMC medium of each group showed that the number of particles secreted by CD137 activation cells was significantly higher than the control group(P<0.05).The number of particles secreted by Rab7 lentivirus interference group was significantly lower than CD137 activation group(P<0.05).The expression of Hsc70 protein in exosomes secreted by CD137 activation group was higher than that in the control group(P<0.05);The expression of Hsc70 protein in exosomes secreted by Rab7 lentivirus interference group was lower than that in the CD137 activation group(P<0.05).There was no difference between the lentivirus control group and the CD137 activation group(P>0.05).(5)Expression of autophagy-related proteins in exosomes of VSMC medium in each group: Lc3 ? protein expression was detected in exosomes of each group,and the expression of Lc3 ? protein in exosome of CD137 activation group was higher than that in control group(P<0.05),the expression of LC3 ? protein in exosome of Rab7 lentivirus interference group was lower than that in CD137 activation group(P<0.05).There was no difference between the lentivirus control group and the CD137 activation group(P>0.05).ConclusionThe CD137-CD137 L signaling may influence the secretion of exosomes steming from mouse vascular smooth muscle cells(VSMCs)through autophagy mediated by Rab7 molecule.