Macrophage CD147 Signaling Mediates The Transformation Of Vascular Smooth Muscle Cells To Synthetic Phenotypes Via MTOR

Objective1.To clarify the expression of CD147 in the process of atherosclerosis and the effect of macrophage CD147 signaling on plaque formation and the phenomenon of transformation of vascular smooth muscle to synthetic phenotype;2.To clarify whether the CD147 signal of intervening macrophages mediates the conversion of vascular smooth muscle cells to a synthetic phenotype through the mTOR signaling pathway in cell experiments.Methods1.In animal experiment,the expression of CD147 in the mouse aortic plaque group and the control group was detected via Western blot and immunofluorescence.2.The immunohistochemical and immunofluorescence experiments were conducted to observe the CD147 protein in the aortic smooth muscle of mice in the plaque group and the control group,as well as the synthetic proteins related to smooth muscle phenotype,including Osteoptoin（OPN）,Calcineruin（Ca N）,and contraction proteins,including Alpha smooth muscle actin.-SMA),myocardial myosin（Smooth muscle myosin heavy chain,SM-MHC）expression.3.In the immunofluorescence double-staining experiment,the blood vessels were divided into（1）control group and（2）plaque group.The macrophage marker protein F4/80 was co-stained with CD147 to clarify the accumulation of macrophages in atherosclerotic plaques as well as the expression of CD147 derived from macrophages as the plaques progressed.4.In cell experiment,the small interfering RNA technology is used to observe the changes of phenotypic related proteins in Vascular smooth muscle cells（VSMC）after the silent expression of macrophage CD147.5.the cell experiments were divided into:（1）control group（VSMC was planted in the lower chamber）;（2）control group with CD147 signal（recombinant CD147protein was added in the upper chamber and VSMC was planted in the lower chamber）;（3）control group with macrophages（macrophages were planted in the upper chamber and VSMC was planted in the lower chamber）;（4）macrophage CD147 signal excitation group（macrophages were planted in the upper chamber+recombinant CD 147 protein and VSMC was planted in the lower chamber）;（5）macrophage CD147 signal inhibition group（upper chamber planted with macrophages+recombinant CD147 protein+CD147 si RNA,and lower chamber planted with VSMC）,Western blot,scratch test,Transwell,and EDU test were performed to detect the activation and silence of CD147 signal of macrophages,as well as the proliferation and migration of smooth muscle cells and the expression of phenotype-related proteins.6.In cell experiment,by further inhibiting mTOR signaling pathway,the experiments were divided into:（1）Agonist-CD 147mΦ-VSMC（macrophages planted in the upper chamber+CD147 recombinant protein,and smooth muscle cells planted in the lower chamber）;（2）Agonist-CD 147-Anti-mTOR-mΦ-VSMC（macrophages planted in the upper chamber+CD147 recombinant protein,and smooth muscle cells+mTOR inhibitor planted in the lower chamber）.Using Western blot,scratch,Transwell,and EDU experiments,we observed the expression of CD147 protein,synthetic protein and contractile protein in smooth muscle cells of each group and the migration and proliferation of cells in each group to determine whether mTOR affects phenotypic transformation of smooth muscle cells.Result1.In the animal model,C57BL/6 mice fed a normal diet had no obvious aortic plaques,CD147 protein（p<0.01）and synthetic protein OPN（p<0.01）,Ca N（p<0.05）expression Lower,higher contractile protein expressionα-SMA（p<0.01）,SM-MHC（p<0.01）;high-fat fed Apo E^-/-mice had larger aortic plaque area,CD147 protein and synthetic The expression of protein is higher,and the expression of contractile protein is opposite to the former;with the progress of atherosclerosis,the expression of F4/80（p<0.01）and CD147（p<0.05）in plaques gradually increases.2.In cell experiments,after using recombinant protein to stimulate CD147,the migration and proliferation ability of smooth muscle cells were significantly increased,and the expression of synthetic proteins was increased.After co-culture of macrophages and smooth muscle cells,the synthetic proteins OPN and Ca N（all p<0.01）expression increased more significantly,and the expression of contractile proteinsα-SMA and SM-MHC（both p<0.05）decreased significantly.After inhibiting the mTOR signal,compared with before,the migration and proliferation ability of smooth muscle cells and the expression of synthetic proteins decreased,while the expression of contractile proteins increased.Conclusion1.As atherosclerotic plaques progress,the accumulation of macrophages in the plaques and the expression of macrophage-derived CD147 increase,and vascular smooth muscle transforms from contractile type to synthetic type.2.After co-culture of macrophages and smooth muscle cells,activation of macrophage CD147 signal may mediate the transformation of vascular smooth muscle cells from contractile phenotype to synthetic phenotype through mTOR signaling pathway.