Mitochondrial Reactive Oxygen Species Mediate Chlamydia Pneumoniae Infection-induced Vascular Smooth Muscle Cell Migration And Atherosclerotic Lesion Formation

Objective:Using Apo E^-/-and Apo E^-/-TLR2^-/-mouse models of Chlamydia pneumoniae（C.pneumoniae）infection-induced atherosclerosis and primary vascular smooth muscle cell（VSMC）infected with C.pneumoniae in vitro,to observe the role of C.pneumoniae infection in mitochondrial function,to confirm that Toll-like receptor 2（TLR2）,mitochondrial reactive oxygen species（mt ROS）,Jun B proto-oncogene（Jun B）,FBJ osteosarcoma phase oncogene related antigen 1（Fra-1）and matrix metalloproteinase 2（MMP2）participated in C.pneumoniae infection-induced VSMC migration and atherosclerotic lesion formation,to explored the interaction between Jun B and Fra-1 and the regulatory effect of Jun B/Fra-1 on MMP2 in this process,to unveil the molecular mechanism of C.pneumoniae infection-promoted VSMC migration and atherosclerotic lesion formation.Methods:Using Apo E^-/- mice fed high-fat diet and infected with C.pneumoniae to observe the changes of the area of atherosclerotic lesion and the content of lipid and VSMCs in the lesions;Using the VSMC infected with C.pneumoniae model to analyze the changes of protein by Mass Spectrometry in vitro,basing on the results of Mass Spectrometry,analyzed further changes of mitochondrial copy number,mitochondrial morphology,mitochondrial membrane potential and the content of mt ROS;Using Wound healing and Transwell experiments to observe the effect of mt ROS on C.pneumoniae infection-induced VSMC migration after scavenging mt ROS by Mito-TEMPO;Analyzed protein AP-1 and MMP2 in VSMC infected with C.pneumoniae by Mass Spectrometry and confirmed by Western blot;Observed the interaction between Jun B and Fra-1 in the infected VSMC by Immunoprecipitation;Using small interference RNA（si RNA）to silence the expression of Jun B,Fra-1 and MMP2 respectively to detect the expression of protein MMP2 by Western blot,using Wound healing and Transwell experiment to observe the effect of Jun B,Fra-1 and MMP2 on the migration of VSMC induced by C.pneumoniae infection;Detected the expression of protein Jun B,Fra-1 and MMP2 by Western blot after scavenging mt ROS by Mito-TEMPO;Using Apo E^-/-TLR2^-/-mice fed high-fat diet and infected with C.pneumoniae model to observe the changes of area of atherosclerotic lesion,the content of lipid and VSMCs in the lesions.Decreasing the expression of protein TLR2 by si RNA,observed the content of mt ROS and the expression of pro Jun B,Fra-1 and MMP2 after VSMC infected with C.pneumoniaeResults:Comparing with the group of mock infection,the area of atherosclerotic lesion and the content of lipid and VSMCs in the lesion in Apo E^-/-mice infected with C.pneumoniae significantly increased.In the infected VSMC model in vitro,C.pneumoniae infection mainly impaired on the function of oxidative phosphorylation and reduced the mitochondrial copy number,shortened the length of mitochondria,increased mitochondrial membrane potential and increased the content of mt ROS.The expression of protein Jun B,Fra-1 and MMP2 significantly increased after C.pneumoniae infection,and the interaction between Jun B and Fra-1 obviously enhanced.After scavenging mt ROS,both the increasing expressions of Jun B,Fra-1,MMP2 and the capacity of migration of VSMC induced by C.pneumoniae infection obviously inhibited.After down regulating the expression of Jun B,Fra-1 and MMP2respectively,the expression of MMP2 and the capacity of migration of VSMC decreased significantly.Comparing with Apo E^-/-mice,the area of atherosclerotic lesion and the content of lipid and VSMCs in atherosclerotic lesion significantly decreased in Apo E^-/-TLR2^-/-mice.Using si RNA to inhibit the expression of TLR2,the content of mt ROS and the expression of Jun B,Fra-1 and MMP2 significantly decreased.Conclusion:C.pneumoniae infection may promote VSMC migration and atherosclerotic lesion formation by increase the content of mt ROS through TLR2 to activate the Jun B/Fra-1/MMP2 signaling pathway.