CD137-CD137L Signaling Affects Autophagy Via Lysosomal Damage In Mouse Vascular Smooth Muscle Cells

ObjectiveTo explore whether CD137-CD137 L signaling can regulate the autophagy via damaging lysosomal function in mouse vascular smooth muscle cells(VSMCs).MethodsThe experiment includes two parts.(1)Mouse thoracic aorta VSMCs were obtained by tissue piece adherence method.The cells were divided into three groups: Blank control group,mixed inflammatory factors pre-stimulation group(control group+10ng/ml TNF-?,IL-1?,IFN-? respectively),CD137 agonist group(mixed inflammatory factors pre-stimulation group +recombinant CD137 L protein).Western blot was used to detect the expression levels of lysosomal associated membrane protein 1(LAMP1),cathepsin B(CTSB)and cathepsin D(CTSD)in VSMCs.The expression of lysosome-associated membrane protein 2(LAMP2)in VSMC was measured by immunofluorescence.Acridine orange(AO)staining was used to check the changes of lysosome p H in VSMCs.The lysosome volume in VSMCs were observed by transmission electron microscopy.(2)VSMCs were prepared by the same method.The experiment was divided into four groups: Blank control group,CD137 agonist group [mixed inflammatory factors + CD137 L recombinant protein(final concentration 10?g/ml)],rapamycin group [rapamycin(DMSO dissolution,final concentration 100 n M)pre-stimulation + mixed inflammatory factors + CD137 L recombinant protein(final concentration 10?g/ml)],DMSO group [adding the same amount of dimethylsulfoxide(DMSO)as rapamycin group].)Prestimulation + mixed inflammatory factors + CD137 L recombinant protein(final concentration 10?g/ml)].Western blot is used to detect the expression of autophagy-related proteins LC3 B and p62.Results(1)Western blot showed that after pre-stimulation of mixed inflammatory factors,the expression levels of LAMP1,CTSB and CTSD in VSMCs were not significantly different from those in the control group(P>0.05).Compared with the control group,the protein level of LAMP1(0.38±0.03 vs 1.08±0.06,P<0.05),CTSB(0.33±0.04 vs 0.57±0.03,P<0.05),CTSD(0.37±0.02 vs 0.90±0.02,P<0.05)in agonist-CD137 group was significantly decreased.(2)Immunofluorescence showed that there was no significant difference in the total number of red fluorescent spots in VSMC between the mixed inflammatory factors pretreatment group and the control group(P>0.05).The total number of red fluorescent spots in VSMCs of CD137 signal activation group was more than that of control group [(107.7±6.33)/cell ratio(33.33±3.52)/cell,P<0.05].(3)The results of living cell staining showed that there was no significant difference in the total number of red fluorescent spots in VSMCs between the mixed inflammatory factors pre-stimulation group and the control group(P>0.05).The total number of red fluorescent spots in VSMCs of CD137 signal activation group was less than that of control group [(20.66±3.53)/cell ratio(90.33±8.19)/cell,P<0.05],which indicated that lysosome p H value increased.(4)The diameter of lysosome observed by transmission electron microscopy showed that there was no significant difference in the volume of lysosome in VSMCs between the mixed inflammatory factors pre-stimulation group and the control group(P>0.05).The diameter of lysosome in VSMCs of CD137 signal activation group was larger than that of control group(1370±133.4nm vs.565.3±68.6nm,P<0.05).(5)Western blot showed that the expression levels of p62 and LC3 II in VSMCs were significantly higher than those in control group(1.15±0.06 vs.0.59±0.07,1.06 +0.08 vs.0.54±0.06,P<0.05).There was no significant difference in the expression of p62 and LC3 II protein between DMSO group and CD137 group(P>0.05).The expression levels of p62 and LC3 II protein in VSMCs after rapamycin addition were significantly lower than those in CD137 signal activation group(0.53±0.04 vs.1.15±0.06?0.52±0.05 vs.1.06±0.08,P<0.05).ConclusionsCD137-CD137 L signaling may regulate the autophagy via damaging lysosomal function in mouse vascular smooth muscle cells(VSMCs).